Activation of Different C1 Currents in Xenopus Oocytes by Ca

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Activation of Different C1 Currents in Xenopus Oocytes by Ca Powered By Docstoc
					Activation of Different C1 Currents in XenopusOocytes by Ca Liberated
from Stores and by Capacitative Ca Influx

         H. CRISS HARTZELL
         From the Department of Anatomy and Cell Biology, Emoly University School of Medicine, Atlanta, Georgia 30322-3030


         ABSTRACT Xenopusoocytes are an excellent model system for studying Ca signaling. The purpose of this study
         was to characterize in detail the Ca-activated C1 currents evoked by injection of inositol 1,4,5-trisphosphate (IP3)
         into Xenopusoocytes voltage-clamped with two microelectrodes. Injection of IP3 into Xenopusoocytes activates two
         different Ca-activated C1 currents. Io_1 is stimulated rapidly (within 5 s after IP 3 injection), exhibits time-depen-
         dent activation upon depolarization, a linear instantaneous IV relationship with a reversal potential near F<:I, and
         a curvilinear activation curve with an approximate half-maximal activation voltage of >200 mV. Icl-2Dis stimulated
         slowly after IP.~ injection (half-maximal stimulation occurs ~ 3 min after injection). ICI_2Dhas a strongly outwardly
         rectifying instantaneous IV relationship with a reversal potential near Eel and is activated by hyperpolarization
         with a half-maximal activation voltage of - 1 0 5 mV. ICI_2Dc a n n o t be activated by Ca released from stores but is acti-
         vated by Ca influx. In contrast, Icl_1 c a n be stimulated by Ca released from intracellular Ca stores. It can also be
         stimulated by Ca influx through store-operated channels if the Ca driving force is increased by a hyperpolarization
         immediately before the depolarization that gates Icl_1 channels. The description of two currents activated by influx




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         and Ca release from stores provides new insights into and questions about the regulation of Ca in Xenopusoocytes.
         Key words: Xenopusoocytes 9 inositol phosphate 9 CI channels 9 calcium 9 Ca channels

INTRODUCTION                                                               1989; Putney, 1992). T h e Ca influx is t h o u g h t to be
                                                                           stimulated by "depletion" of internal stores because a
T h e concentration of cytosolic free Ca ([Ca]i) regu-
                                                                           similar Ca influx is stimulated by other interventions
lates m a n y physiological processes. These processes are
                                                                           that lower store Ca. Ca-ATPase inhibitors thapsigargin,
controlled by receptors that activate Ca release f r o m in-
                                                                           cyclopiazonic acid, and B H Q as well as intracellular
tracellular stores or increase Ca influx f r o m the extra-
                                                                           bis (o-aminophenoxy) ethane-N,N,N',N'-tetracetic acid
cellular space (Tsien and Tsien, 1990; Pozzan et al.,
                                                                           (BAPTA) deplete Ca stores passively by preventing re-
1994; Clapham, 1995). Ca is mobilized f r o m internal
                                                                           uptake o f Ca that leaks out (see Pozzan et aI., 1994).
stores by inositol 1,4,5-trisphosphate (IP.~) 1 p r o d u c e d
                                                                           T h e Ca i o n o p h o r e ionomycin at low concentrations
by activation of m a n y G - p r o t e i n - c o u p l e d and tyrosine
                                                                           can deplete stores and activate capacitative Ca entry
k i n a s e - c o u p l e d receptors that stimulate phospholipase
                                                                           without directly affecting the plasma m e m b r a n e (Mor-
C. Release of Ca from internal stores is often followed
                                                                           gan and Jacob, 1994). Store-operated Ca channels are
by a sustained influx of extracellular Ca (Putney, 1990;
                                                                           not o p e n e d directly by IP3 or Ca, because Ca ATPase in-
Meldolesi et al., 1991; Putney, 1992; Putney, 1993; Faso-
                                                                           hibitors and ionomycin do not stimulate IP3 produc-
lato et al., 1994). This influx ("capacitative Ca entry") is
                                                                           tion, and BAPTA prevents elevation of cytosolic Ca.
m e d i a t e d by store-operated Ca channels (SOCCs) in
the p l a s m a l e m m a that are apparently controlled by the
                                                                              Xenopus oocytes have b e e n a very useful m o d e l system
                                                                           for studying calcium signaling partly because these ceils
level of Ca in the internal store.
                                                                           express Ca-activated C1 channels that can be used as
    T h e activation of a Ca influx pathway subsequent to
                                                                           real-time indicators of cytosolic Ca concentration (Das-
Ca liberation f r o m stores has b e e n d e m o n s t r a t e d by
                                                                           cal, 1987) and partly because their large size facilitates
showing that activation of PLC-coupled receptors pro-
                                                                           the study of spatial and t e m p o r a l changes in cytosolic
duces only a transient increase in cytosolic Ca concen-
                                                                           Ca concentrations (Girard and Clapham, 1993; Lech-
tration w h e n the cell is b a t h e d in zero Ca solution, but
                                                                           leiter and Clapham, 1992). For the past year, we have
addition of extracellular Ca results in a large sustained
                                                                           b e e n using Xenopus oocytes as a heterologous expres-
increase in intracellular Ca ( T a k e m u r a and Putney,
                                                                           sion system for putative store-operated Ca channels
                                                                           and have used the e n d o g e n o u s Ca-activated C1 currents
                                                                           as an indirect assay of cytosolic Ca concentrations.
Address correspondence to H. Criss Hartzell, Department of Anat-           These C1 currents have b e e n studied extensively by oth-
omy and Ceil Biology, Emory University School of Medicine, Atlanta,
                                                                           ers (see Dascal [1987] and references in DISCUSSION).
Georgia 30322-3030_ Fax: 404-727-6256; E-mail: criss@anatomy.
emory.edu                                                                  However, we f o u n d that the behavior of these currents
  IAbbreviations used in this paper: IP:~, inositol 1,4,5-trisphosphate,   was m o r e c o m p l e x than we anticipated and f o u n d it
BAPTA, bis(0-aminophenoxy)ethane-N,N,N',N'-tetracetic acid.                necessary to undertake a m o r e detailed, quantitative

                                  157     J. GEN. PHYSIOL.9 The Rockefeller University Press 9 0022-1295/96/09/157/19 $2.00
                                          Volume 108 September 1996 157-175
analysis of them in order to interpret our results with                                       normal Ringer with no added calcium containing 2 m g / m l colla-
heterologously expressed channels. In our analysis, we                                        genase type IA (Sigma Chemical Co., St. Louis, MO) for 2 h at
                                                                                              room temperature. The oocytes were extensively rinsed and
h a v e f o u n d t h a t t h e r e a r e t w o d i s t i n c t C a - a c t i v a t e d C1
                                                                                              placed in L15 m e d i u m (Gibco BRL, Gaithersburg, MD) a n d
c u r r e n t s i n Xenopus o o c y t e s a n d t h a t o n e c u r r e n t is acti-          stored at 18~ Oocytes were usually used between 1 and 6 d after
vated preferentially by Ca influx through store-oper-                                         isolation.
ated channels and that the other can be activated both
b y C a i n f l u x a n d b y C a r e l e a s e f r o m i n t e r n a l s t o r e s . Be-     Display and Analysis of Data
cause these currents are dually regulated by voltage                                          For display of the figures, current transients during voltage steps
a n d b y Ca, i n t e r p r e t a t i o n o f e f f e c t s o f e l e v a t i o n o f cyto-   were either blanked for 4 ms (e.g., see Fig. 2 B) or 4 ms of the
solic Ca can be complicated.                                                                  data was replaced with the value of the current immediately be-
                                                                                              fore the voltage step (e.g., see Fig. 3 B). Data points are the mean
                                                                                              a n d error bars are _+SEM. Each current-voltage and activation
METHODS

Electrophysiological Methods
Xenopus oocytes were voltage-clamped with two-microelectrodes                                              _35mYj                +20mV
using a GeneClamp 500 (Axon Instruments, Foster City, CA).                                     A                                                         [             -120mY
Electrodes were filled with 3 M KC1 a n d had resistances of 1-2
MII. Typically, the m e m b r a n e was held at - 3 5 mV, a n d voltage                                    2000
steps were applied as described in the text. Stimulation a n d data
acquisition were controlled by PClamp 6.01 (Axon Instruments)
~4a a Digidata 1200 A / D - D / A converter (Axon Instruments) and
                                                                                                            1000




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a Gateway P5-90 c o m p u t e r (Intel Pentium, 90 MHz). During re-
cording, the oocyte was superfused with normal Ringer solution.
The bath c h a m b e r volume was ~ 3 0 0 ~1 a n d was superfused at a                               <
                                                                                                     r
rate of 2 m l / m i n unless the composition of the solution was be-                                  v                 a
ing changed, in which case superfusion rates as high as 15 ml per                                     t-
                                                                                                                                                         r
                                                                                                                                                                          (a) controt
min were used. W h e n the composition of the bath was to be
                                                                                                     O
changed, the superfusion rate of the control solution was in-
creased for several minutes before changing to the new solution                                            -1000
at the same flow rate. Experiments were performed at room tem-
                                                                                                                                                                        (c) 8m after IP3
perature (22-26~
                                                                                                           -2000                  I
Microinjection                                                                                                      o            5oo                lOOO               15'oo        20'00
Oocytes were injected with IP~ or BAPTA using a Nanoject Auto-                                                                               Time (ms)
matic Oocyte Injector ( D r u m m o n d Scientific Co., Broomall, PA).
The injection pipet was pulled from glass capillary tubing in a
m a n n e r similar to the recording electrodes and then broken so                             B           2000-
                                                                                                                                 IP3    b
that it had a beveled tip with an inside diameter of < 2 0 Ixm. Typ-
ically, 4.6 nl of a 10 mM solution Is in H20 was injected to give a
calculated oocyte concentration of ~ 5 0 p~M. The Ca concentra-
tion in this solution was not buffered, but injection of H 2 0 pro-                                        1000
duced no change in m e m b r a n e current. Usually we injected 23                                                                    l~:~                              I~ = la.1        c
nl of a 50 mM solution of K4'BAPTA in water to give a final calcu-                                    <
lated concentration in the oocyte of 1 mM. The injection pipets
                                                                                                      e-
were usually left impaled in the oocyte for the duration of the ex-
periment, unless the oocyte was injected with two different solu-
                                                                                                      o
tions, in which case the first pipet was withdrawn before the sec-
o n d pipet was inserted.
                                                                                                           -1000
Solutions
Normal Ringer consisted of 123 mM NaC1, 2.5 mM KCI, 1.8 mM                                                          i        i                i                    i       i             L
                                                                                                                   0        100              200               300        400           500
CaCI,2, 1.8 mM MgCI 2, 10 mM HEPES, pH 7.4, 5 mM glucose, 5 mM
sodium pyruvate. Zero Ca Ringer was the same except CaCI 2 was                                                                                    Time       (s)
omitted, MgCI~ was increased to 5 mM, a n d 0.1 mM EGTA was
added. For 10 mM Ca Ringer, CaC12 was increased to 10mM.                                      FIGURE 1. Effects of intracellular injection of IP~ on ionic cur-
                                                                                              rents in Xenopusoocytes. (A) Current responses to 1-s voltage step
Harvesting Eggs                                                                               to +20 mV followed by 1-s step to - 1 2 0 mV from a holding poten-
                                                                                              tial of - 3 5 inV. Trace a is before, trace b 30 s after, and trace c 8
Stage V-VI oocytes were harvested from adult Xenopus laevis fe-                               min after IP 3 injection. (B) Time course of change in current at
males (Xenopus I) as described by Dascal (Dascal, 1987). Xenopus                              end of +20 mV pulse (I9~5) and at end of - 120 mV pulse (12(xx)) a s
were anesthetized by immersion in Tricaine (1.5 g/liter). Ovar-                               a function of time after injection of IP 3. IP3 was injected at the ar-
ian follicles were removed, cut into small pieces, and digested in                            row. The letters a, b, and c correspond to the traces in A.

                                           158         DifferentC1 Curr~ts Activated by Ca Influx and Ca Stores
                                               +lOOmY                               +80mV            curve is the average of three to six different oocytes. For current-
A                                                                                                    voltage relationships, the raw data was averaged. For the activa-
                                                                                                     tion curves, the data was normalized such that the current at a
            12OOO                                                   ~-120rnV                         particular voltage was set as 1.0. Mathematical fits were per-
                                                                                                     formed using an iterative Levenberg-Marquardt algorithm. Un-
             9OOO                                                                                    less noted, the interval between stimuli was 10-20 s. In tail cur-
                                                                                                     rent analysis, one would like to be able to measure the current
             6OOO
                                control                                                              immediately after the voltage step, but because of the capacita-
                                                                                                     tive transient, we were not able to measure the instantaneous cur-
             3O0O                                                                                    rent sooner than 4 ms after the step. However, this error seems
  "E                                                                                                 unlikely to contribute significantly to our conclusions because ex-
   .=               0                                                                                periments where we carefully subtracted capacitative transients
                                                                                                     provided the same IV curves as the ones where current was mea-
  o         -3oooi                                                                                   sured at 4 ms.

            -6ooo I                                                                                  R E S U L T S


            .~oooi                         I                 I      i        |      ,        I
                                                                                                     IP3 Injection Activates Two Currents with Different Kinetics
                                         200             400                600         800

                                                         Time (ms)                                   Fig. 1 s h o w s t h e e f f e c t o f i n j e c t i n g 4.6 nl o f 10 m M IP3
                                                                                                     to p r o d u c e a c a l c u l a t e d i n t r a o o c y t e IP 3 c o n c e n t r a t i o n
 B          12000
                                                                                                     o f " - ' 5 0 I~M. T h e s t a n d a r d v o l t a g e p r o t o c o l f o r t h e s e ex-
                                 IP3
             9000                                                                                    p e r i m e n t s was a 1-s d u r a t i o n p u l s e f r o m a - 3 5 m V
                                                                                                     h o l d i n g p o t e n t i a l to + 2 0 m V f o l l o w e d by a 1-s d u r a t i o n




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             6000
                                                                                                     p u l s e to - 1 2 0 m V . T h e v o l t a g e p u l s e u n d e r b a s a l c o n -
             3000                                                                                    ditions produced only small time-independent currents
   e-
  "E                                                                                                 (Fig. 1 A, a). H o w e v e r , i m m e d i a t e l y a f t e r i n j e c t i n g IP 3,
                    0                                                                                a n o u t w a r d c u r r e n t was e l i c i t e d by t h e + 2 0 m V p u l s e
   1--
  (3                                                                                                 (Fig. 1 A, b). T h i s c u r r e n t e x h i b i t e d two c o m p o n e n t s ,
            -3000
                                                                                                     a n i n s t a n t a n e o u s i n c r e a s e f o l l o w e d by a slowly d e v e l o p -
            -6000                                                                                    i n g o u t w a r d c u r r e n t . As s h o w n b e l o w , t h e s e two c o m p o -
                                                                                                     n e n t s w e r e a t t r i b u t e d to t h e s a m e c u r r e n t : t h e i n s t a n t a -
            -9000           I        i     I         i       I               I              I    ,
                                                                                                     n e o u s c o m p o n e n t r e f l e c t e d i n c r e a s e d c u r r e n t ( d u e to
                            0            200             400                600         800
                                                                                                     the increased driving force) through channels that
                                                         Time (ms)
                                                                                                     w e r e a l r e a d y o p e n at - 3 5 m V , a n d t h e t i m e - d e p e n d e n t
                                                                                                     c o m p o n e n t r e f l e c t e d v o l t a g e - d e p e n d e n t o p e n i n g o f ad-
                                                                                                     d i t i o n a l c h a n n e l s . T h e t i m e - d e p e n d e n t c o m p o n e n t was
        C                                                    6000
                                                                                                     w e l l - f i t t e d w i t h two e x p o n e n t i a l s w i t h "r = 28 a n d 274
                                                                                                     ms at + 20 mV. U p o n r e p o l a r i z a t i o n to - 1 2 0 m V , a tail
                                                                                                     c u r r e n t was o b s e r v e d t h a t r a p i d l y b u t i n c o m p l e t e l y in-
                        IcH Steady-state IV
                                                                                                     a c t i v a t e d w i t h -r = 17 a n d 67 ms. T h i s tail c u r r e n t was
                                                             4000
                                                                                                     d u e to t h e v o l t a g e - d e p e n d e n t c l o s i n g o f c h a n n e l s t h a t
                                                                                                     w e r e o p e n at + 2 0 mV. T h i s o u t w a r d c u r r e n t a n d its as-
                                                                                                     s o c i a t e d tail c u r r e n t was n a m e d Ic]_1 W e b e l i e v e t h a t this
                                                             2000
                                                                                                     c u r r e n t is a C a - a c t i v a t e d C1 c u r r e n t t h a t is i n d u c e d by
                                                                                                     C a r e l e a s e d f r o m i n t r a c e l l u l a r s t o r e s by t h e i n j e c t e d
                    -100                       -60                      J               5O
                                                                                                     IP3. O u r r e a s o n s f o r this c o n c l u s i o n a r e b a s e d o n t h e
            9~          ~       ~,       -~- i ~ . ~ - , ~                              t
                                                                                                     o b s e r v a t i o n s t h a t IcH (a) was b l o c k e d by 0.5 m M niflu-
                                                                                  Voltage (mV)
                                                                                                     m i c a c i d (a C a - a c t i v a t e d CI c h a n n e l b l o c k e r [ W h i t e a n d
FIGURE 2. Steady-state IV relationship of Ict-v The oocyte was                                       Aylwin, 1 9 9 0 ] ) , (b) h a d a r e v e r s a l p o t e n t i a l n e a r t h e C1
stepped from - 6 0 mV to different potentials between - 4 0 and                                      e q u i l i b r i u m p o t e n t i a l , a n d (c) its i n d u c t i o n was i n d e -
+100 mV for 500 ms followed by a step to - 1 2 0 mV for 50 ms and                                    p e n d e n t o f e x t r a c e l l u l a r C a a n d was u n a f f e c t e d by ex-
a 500 ms step to +80 mV as shown. (A) Steady-state IV traces be-
                                                                                                     t r a c e l l u l a r La, Ba, o r M n . T h e s e d a t a a r e s h o w n b e l o w .
fore IP 3 injection. (B) Steady-state IV traces after IP 3 injection. (C)
Average steady-state IV relationship for six oocytes before (open cir-
cles) and after (solid squares) IP 3 injection. The currents at the end
of the first voltage pulses (at 495 ms) were plotted vs. the test po-                                - 120 mV and the test voltage steps were from - 120 to + 80. In the
tential. This curve was constructed from two different voltage pro-                                  voltage range where these two protocols overlapped ( - 4 0 to +80
tocols. The first protocol was identical to the one shown in A and                                   mV) the data points were identical within <10%, so the data were
B. The other was similar, except that the holding potential was                                      pooled.

                                                         159                HARTZELL
                               I
                                           +56       mV                                                                              the f o r m I = I 0 + A*[1 - e x p ( - x / ' r ) ] P w h e r e p : 2 a n d
A               -3smvI
                400O
                                                                                                          I b               -94 mV
                                                                                                                                     "r = 289 ms. This inward c u r r e n t was n o t a c c o m p a n i e d
                                                                                                                                     by a significant o u t w a r d c u r r e n t at + 2 0 mV using this
                                                                                                                                     protocol. This inward c u r r e n t was n a m e d Icy). This cur-
                20oo                   control
                                                                                                                                     r e n t was also a Ca-activated C1 current, as it was b l o c k e d
                                                                                                                                     by 0.5 m M niflumic acid a n d h a d a reversal potential
                                                                                                                                     that c o i n c i d e d with the C1 equilibrium potential. How-
                                                                                                                                     ever, this c u r r e n t r e q u i r e d influx o f extracellular Ca to
        'E                                                                                                                           be activated as described below.
                           o       h
         :3
        (J                     /                                                                                                         T h e time course o f d e v e l o p m e n t a n d decay o f Ic~_
                                                                                                                                     a n d Ic1_ are shown in Fig. 1 B. In this plot, IcH was mea-
                                                                                                                                               z
                                                                                                                                                                                                               ~

                                                                                                                                     sured as the c u r r e n t at the e n d o f the + 2 0 mV pulse,
               -2OO0 f ,               .         ,       .       ,        .   ,     .        ,
                                                                                                                                     a n d I(;1.2 was m e a s u r e d at the e n d o f the - 1 2 0 mV
                                                                                                                                     pulse. IcH d e v e l o p e d fully within 30 s after injection o f
                                                                                                                                     IP3 a n d t h e n d e c l i n e d nearly to baseline in ~ 1 . 5 min.
                                                                                                                                          z
                                                                                                                                     Ic~_ d e v e l o p e d slowly a n d was n o t maximally activated
 B             4 0 0 0 -



                                                                                                                                     until > 5 min after IP.~ injection.

               2000 -                                                                                                                It:l_1 Is a Cl Current with a Linear Instantaneous
                                                                                                                                     Current-Voltage Relationship




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        r
                                                                                                                                     Steady4tate I V relationship. T h e "steady-state" IV curve
        ,...
                                                                                                                                     for Ic~ was d e t e r m i n e d using the p r o t o c o l shown in
        0
                                                                                                                                     Fig. 2, A a n d B. T h e cell was d e p o l a r i z e d to different
                                                                                                                                     c o m m a n d potentials for 500 ms f r o m a h o l d i n g poten-
               -2000
                                                                                                                                     tial o f - 3 5 mV before (Fig. 2 A) or immediately after
                                                     =       9        i         9       r        9        i         9        i
                                                                                                                                     (Fig. 2 B) injection o f IP~. T h e a m p l i t u d e o f the out-
                                                 500                 1000           1500                 2000               2500
                                                                                                                                     ward c u r r e n t at the e n d o f the c o m m a n d pulse was
                                                                     Time (ms)




                                                                                                                                             1.2.
    C
                                   Ic~,~Instantaneous IV                      6___ t             T ~ /                  ~
                                                                                                                                             1.0.
                                                                                                                                                             Ict.1 Activati
                                   Voltage (mV)
                                                                                                                                             0.8.
                                                                                                     9          i


                                                                                                                                     <
                                                                                                                                     e-      0.6-


                                                                                                                                     t..     0.4.
                                                                                                                                     O
                                                                                                                                             0.2.
FIGURE 3. Instantaneous 1V relationship of Icl_I. The oocyte was
stepped from - 3 5 to +60 mV for 500 ms followed by short repo-                                                                              0.0.
larizations of 50 ms to different test potentials between +50 and
                                                                                                                                                            ,             ,          ,        ~
- 100 mV. (A) IV traces before IP3 injection. (B) IV traces after IP:~                                                                         -150       -100          -50          0       6         1 0
injection. (C) Average instantaneous IV relationship for four oo-
cytes determined as shown in B. The current was measured at 4 ms                                                                                                              Voltage (mY)
after the start of the test pulses.
                                                                                                                                     FIGURE 4. Activation curve of IcH. The activation curve was deter-
                                                                                                                                                                                                x
                                                                                                                                     mined using the protocol shown in Fig. 2, A and B. Ic]_ was acti-
                                                                                                                                     vated by 500-ms duration prepulses to different potentials. The ac-
                                                                                                                                     tivation curve was then determined by measuring the current 4 ms
    O v e r t h e n e x t s e v e r a l m i n u t e s , t h e Ic~_l o u t w a r d c u r -                                            after hyperpolarizing to a test potential of - 120 mV and ploued as
r e n t a n d its i n w a r d tail c u r r e n t b e c a m e s m a l l e r i n a m -                                                 a function of the prepulse potential. As described in the legend to
                                                                                                                                     Fig. 2 C, this euwe was constructed from two slightly different pro-
p l i t u d e . C o n c u r r e n t w i t h t h e d e c r e a s e i n [eL_l, a t i m e -
                                                                                                                                     tocols (holding potentials of - 6 0 mV or - 1 2 0 mV). In the region
d e p e n d e n t i n w a r d c u r r e n t d e v e l o p e d in r e s p o n s e to                                                  where these protocols overlapped, the curves were identical within
t h e v o l t a g e s t e p t o - 1 2 0 inV. T h i s c u r r e n t e x h i b i t e d a                                               <10%. The data were fitted to the Boltzmann equation: I = 7.94/
s i g m o i d o n s e t a n d was f i t t e d w e l l b y a n e x p o n e n t i a l o f                                              I1 + exp (v-226)/64] + 7.96.

                                                                          160               Different Cl Currents Activated by Ca Influx and Ca Stores
                                                                                                                  +80    mV
                                                 +20   mV

A          -~m~
                                                                                                                                                B
        1500                                                                                                      -140   mV
                                                                                                                                                     1500
                                Control                                                                                                                                               IP 3                                                        ~._
        1000                                                                                                                                         1000

         500                                                                                                                                          500       ,.,,,,       , ....   .              .   .       .   .   .   .   .   .   .    .   .    .   .        .   .       .       .        .       .       .        .   .




<           o       L .,,              , ,
                                                                                                                                            <           0
i-
v
                                                                                                                                                                                                                                                  .'
                                                                                                                                                                                                                                                  ,
                                                                                                                                                                                                                                                            ,
                                                                                                                                                                                                                                                                i
                                                                                                                                                                                                                                                                                    I
                                                                                                                                                                                                                                                                                            .,       '
                                                                                                                                                                                                                                                                                                             '
                                                                                                                                                                                                                                                                                                                     -
                                                                                                                                                                                                                                                                                                                         --
                                                                                                                                                                                                                                                                                                                              9
                                                                                                                                                                                                                                                                                                                                  -
                                                                                                                                                                                                                                                                                                                                      '
                                                                                                                                                                                                                                                                                                                                          '    "       .
                                                                                                                                                                                                                                                                                                                                              . . . . . . .
                                                                                                                                                                                                                                                                                                                                                               "


                                                                                                                                                                                                                                                  t
E        -500                                                                                                                               E        -500                                                                                         f ....                    ,                                            . . . . . . . . . . . . .

 L.-



0       -I000                                                                                                                                       -1000

        -1500                                                                                                                                       -1500

        -2000                                                                                                                                       -2000
                                                        '
                                                       500                        10'00                '          1500              2000                                          '            5+0                       '                   10'00                                  '                                    1 '00                                20'00
                                                                     Time (ms)                                                                                                                                               Time (ms)

    C   1500                                                                                                                                            D                                                                                                  1500-~                                                                                      T
                                             IP3+Mn                                                                                                                                   Ic[_2Steady-stateIV
        1000                                                                                                                                                                                                                                               1000t                                                                                       ~ / ~ _ IP3
                                                                                        I


                                                                                        t,                                                            Voltage (mV)                                                                                                  500 t                                                     ~
                                                                                                                                                                                                                                                                                                                          -;v., - ~                                'P3+Mn




                                                                                                                                                                                                                                                                                                                                                                             Downloaded from www.jgp.org on February 20, 2005
         500
                .   l L:55'.,   !r!&    .5 '   L, L.    5    .   :   2!i   J!Ul   Li5       . . . . . . . . . .               .,,
                                                                                                                                                        -150                          -100                                       -50                   ~                                                                  "-Y-                ~.~/~                Control
~'          0 iM                                                                        J        ~                                                          *            '                a.                 ,                                                  -                                                                                  i


                                                                                                                                                                                                                                                                                                                                              ,o
E        -500

0       -1000                                                                                                                                                                                                                                              -lOOO-


        -1500                                                                                                                                                                                                                                              _15o02
                                                                                                                                                                                                                                                                                                                                  Q.

        -2000                                                                                                                                                                                                                                              -2000
                                         '                                        10'00                 '         15'00             20'00
                                                                     Time (ms)
FIGURE 5. Steady-state IV relationship for Icl,2. Steady state IV relationship was d e t e r m i n e d by giving a 1-s duration pulse from a h o l d i n g
potential o f - 3 5 to +20 mV followed by a 1-s test pulse to different potentials. (A) IV traces before IP s injection. (B) IV traces ~ 1 5 min af-
ter IP3 injection. (C) IV traces after IP 3 injection a n d addition o f 1 m M MnCI 2 to the bath. (D) Average steady-state IV relationship from
four cells m e a s u r e d as shown in A-C. T h e c u r r e n t at 1,995 ms was plotted vs. test potential. Before IP3 injection (solid squares), ~ 1 5 min af-
ter IP~ injection (open circles), a n d after IP 3 injection with Mn a d d e d to the bath (open triangles).




plotted vs. the c o m m a n d potential to give the steady-                                                                                           T h e current (I) plotted in Fig. 2 Cis equal to:
state IV curve (Fig. 2 C). T h e curve in Fig. 2 C is the av-
erage of four oocytes, but each individual oocyte had                                                                                                                                          I = N. Po. ~. (Em-E, ev),
an 1V curve with the same shape. T h e IcH steady-state                                                                                             where N is the n u m b e r of available channels, Po is the
IV curve rectified strongly in the outward direction                                                                                                channel o p e n probability, ~/is single channel conduc-
(Fig. 2 C). A p r o b l e m we e n c o u n t e r e d in p e r f o r m i n g                                                                         tance, E m is m e m b r a n e potential, and Erev is the reversal
these e x p e r i m e n t s was that the amplitude of IcH                                                                                           potential of the current. To d e t e r m i n e which of these
c h a n g e d quickly after injecting IP3 (Fig. 1 B). To mea-                                                                                       terms might contribute to the rectification, we mea-
sure the IV curve quickly while IcH was not changing                                                                                                sured the instantaneous IV curve of Icl. 1 a s shown in
significantly, pulses were given at 2-s intervals. Further,                                                                                         Fig. 3.
to verify that the amplitude of Icl-1 had not c h a n g e d                                                                                            Instantaneous IV relationship. To measure the IV rela-
during the run, the m e m b r a n e was stepped to +80 mV                                                                                           tionship, the m e m b r a n e was stepped to 60 mV for 500
for 500 ms at the end of each trial to verify that It1-1 am-                                                                                        ms to activate Icl. 1 and then was hyperpolarized to dif-
plitude at this given voltage was the same for all trials.                                                                                          ferent test potentials for 50 ms (Fig. 3, A and B). If we
In Fig. 2 B, the amplitude of Icl_l at 950 ms was the same                                                                                          assume that only the driving force (/~n - F~ev) c h a n g e d
for all the trials.                                                                                                                                 instantaneously, the initial amplitude at the onset of

                                                                       161                      HARTZELL
the test pulse is equal to the c u r r e n t t h r o u g h the chan-                 1 s before s t e p p i n g to different test potentials between
nels that were o p e n at the e n d o f the 60-mV c o m m a n d                      - 1 4 0 m V a n d + 8 0 mV for 1 s. T h e c u r r e n t at the e n d
pulse. T h e i n s t a n t a n e o u s c u r r e n t was p l o t t e d vs. the po-   o f the test pulse was plotted vs. the test potential. Un-
tential o f the test pulse. T h e interval between h y p e r p o -                   d e r basal conditions, the cell exhibited a small out-
larizing pulses was adjusted to the m i n i m u m r e q u i r e d                    wardly rectifying c u r r e n t with a slope c o n d u c t a n c e be-
for IcH to r e t u r n to its fully activated a m p l i t u d e (dotted              tween 0 mV a n d - 1 4 0 mV o f ~ 3 ~S (Fig. 5, A a n d D).
horizontal line in Fig. 3 B) b e f o r e the next hyperpolariz-                      15 m i n after injection o f IP3 (Fig. 5 B), the cell exhib-
ing pulse. T h e initial a m p l i t u d e o f the tail c u r r e n t was            ited a tilde ( ~ ) - s h a p e d c u r r e n t voltage relationship:
t h e n plotted vs. the test potential. Before IP 3 injection,                       that is, the c u r r e n t negative to the reversal potential in-
only very small c u r r e n t responses were r e c o r d e d in re-                  wardly rectified a n d the c u r r e n t positive to the reversal
sponse to the voltage pulses (Fig. 3 A). However, after                              potential outwardly rectified (Fig. 5 D). T h e c u r r e n t
IP3 injection, the h y p e r p o l a r i z i n g pulses p r o d u c e d              h a d a reversal potential n e a r the calculated C1 equilib-
large c u r r e n t responses (Fig. 3 B). T h e average instan-
taneous IV curve for Icl.1 r e c o r d e d f r o m f o u r oocytes
(Fig. 3 C) was essentially linear a n d h a d a reversal po-
tential very close to the calculated C1 equilibrium po-
tential ( - 2 0 mV) (Dascal, 1987). T h e fact that the in-                          A
stantaneous IV curve was essentially linear suggested                                                    s,...-.,.,. ,v    ,zoo]
that the rectification o f the steady-state IV was n o t due
to rectification o f c u r r e n t t h r o u g h o p e n channels. This
suggested that v o l t a g e - d e p e n d e n t gating o f the c h a n n e l




                                                                                                                                                              Downloaded from www.jgp.org on February 20, 2005
was responsible for rectification.
    Activation curve. To test this hypothesis, we m e a s u r e d                               46       '     - 1 ; o ~                '   s'o "
the activation curve o f IcH using the same p r o t o c o l
shown in Fig. 2, A a n d B. In this protocol, a different
n u m b e r o f c h a n n e l s were activated by s t e p p i n g to dif-
f e r e n t c o m m a n d potentials between - 4 0 mV a n d + 100
m V for 500 ms. T h e m e m b r a n e was t h e n repolarized to                             Total ~                       -2ooo    (~
a test potential o f - 1 2 0 mV for 50 ms, a n d the initial
a m p l i t u d e o f the tail c u r r e n t was measured. We also
                                                                                                                           -2500
used a similar p r o t o c o l with a larger c o m m a n d p o t e n -
tial r a n g e o f - 120 to 80 mV. Because the a m p l i t u d e o f
the tail c u r r e n t was always m e a s u r e d at the same p o t e n -
tial, E~ - Ere,, was constant. We a s s u m e d that ",/did n o t                        B                                  200
                                                                                                     Voltage (mV)
c h a n g e instantaneously as a f u n c t i o n o f voltage, there-
fore this m e a s u r e m e n t p r e d i c t e d the fraction o f chan-                                                                    50
nels o p e n at different voltages. These data s h o w e d the                                                              -200.
activation o f IcH increased with depolarization, b u t the
c u r r e n t was n o t maximally activated even with com-
                                                                                                                T/          -400
m a n d potentials as high as + 1 0 0 mV (Fig. 4). T h e data                                                _;_/           .6oo    v

in Fig. 4 fitted to the B o l t z m a n n e q u a t i o n suggested
                                                                                                         /1                 -6oo
that the half-activation potential was + 2 2 6 mV, al-                                               _




t h o u g h the precision o f the p a r a m e t e r s derived f r o m                                                               o
                                                                                                  D4(/
this fit s h o u l d be viewed with s o m e skepticism.
    Thus, the steady-state IV o f IcH outwardly rectified
                                                                                             Icl-2 ~ -                     -I000
                                                                                                                           -12oo

because o f the voltage d e p e n d e n c e o f c h a n n e l activa-
tion, b u t the c u r r e n t t h r o u g h the o p e n c h a n n e l s h a d        FIGURE 6. Steady-state IV relationships for tile time-dependent
a p p r o x i m a t e l y a linear current-voltage relationship.                                                                 ~,
                                                                                     and time-independent components of ICl_ The voltage protocol
                                                                                     shown in Fig. 5 was used. (A) Current-voltage relationships for to-
ICl.2 Is a Cl Current with a Strongly Outwardly Rectifying                           tal (open triangles) and time-independent (open squares) component
                                                                                            2.              2
                                                                                     of Icl_ The total Ic~. current was measured as the current at the
Instantaneous I V Relationship                                                       end of the voltage pulse (1,995 ms) as shown in Fig. 5 D (opal cir-
Steady-statelVrelationship. Fig. 5 illustrates the steady-                           cles). The time-independent component was measured as the cur-
                                                                                     rent at 1,020 ms. (B) Current-voltage relationship for the time-
state current-voltage relationships for oocytes b e f o r e                          dependent component of Icl.2 The difference between the current
a n d ~ 1 5 m i n after IP3 injection. T h e m e m b r a n e was                     at 1,995 and 1,020 ms was plotted as a function of the command
s t e p p e d f r o m a h o l d i n g potential o f - 3 5 to + 2 0 mV for            potential.

                                       162       Different CI Currents Activated by Ca Influx and Ca ,Stores
                                                                  +80 m V
  A        -35my                                                                                    B
                             -140 m V                                                              10000
       6000
                                                                                                    8000
                         Control                                                                                         IP 3

       4000                                                                                         6000

                                                                                                    4000
"~     2000
                                                                                                    2000
                                                                                               0
            0                                                                            I               o
                                                                                                    -2000
       -2000             ,       ,        ,           ,       ,      9       ,       .
                0               500                 1000          1500              2000                                     500           1000                 1500
                                              Time (ms)                                                                                 Time (ms)
                                                                                                                                                                                      IP3
 c                                                                                                      D
                       IP3+Mn
       6000                                                                                                          Icl.2Instantaneous
                                                                                                                                      IV               ~[.      6 0 0 0 /




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"~
       4000


       2000                                                                                        Voltage (mY)
                                                                                                                                                       ~
                                                                                                                                                 /
                                                                                                                                                            ~                         IP3+Mn
                                                                                                         -150            -100            -50     /                                   Contro
                t--
                                                                                                                                                                         50
       -2000    I        ,        I
                                500       '         lO'OO         15'oo       '     2o'oo

                                              Time (ms)
                                                     z.
F m u ~ 7. Instantaneous I-V relationship for Icl_ Instantaneous IV relationship was determined by giving a 1-s duration pulse to -140
mV from a holding potential of - 3 5 mV followed by a 1-s test pulse to different potentials. (A) IV traces before IP.~injection. (B) IV traces
~15 min after IP 3 injection. (C) IV traces after IP3 injection with 1 mM MnC12 added to the bath. (D) Average instantaneous current-volt-
age relationship for four oocytes determined as shown in A-C. The current 4 ms after stepping to the test potential (current at 1,004 ms)
was plotted as a function of the test potential. Before IP~ injection (filled circles), after IP3 injection (open squares), and after IP3 injection with
1 mM MnC12 in the bath (filled squares). The slowly developing current during the test pulse is described in more detail in Fig. 11.


r i u m p o t e n t i a l . W h e n 1 m M M n C I 2 was a d d e d to t h e                         t h e d i f f e r e n c e b e t w e e n t h e c u r r e n t a t 1,995 m s a n d t h e
b a t h i n g solution, the t i m e - d e p e n d e n t c o m p o n e n t o f Icl_2                c u r r e n t at 1,020 ms. T h e steady-state t i m e - d e p e n d e n t
was c o m p l e t e l y b l o c k e d . T h e t i m e - i n d e p e n d e n t c u r r e n t        c o m p o n e n t a p p e a r e d to rectify inwardly, w h e r e a s t h e
was p a r t l y r e d u c e d b u t usually r e m a i n e d l a r g e r t h a n be-                current-voltage relationship of the time-independent
f o r e IP 3 i n j e c t i o n (Fig. 5, C a n d D). T h e s e results a r e                        c o m p o n e n t was essentially l i n e a r . W e t e r m t h e s e two
c o n s i s t e n t with t h e s u g g e s t i o n t h a t t h e t i m e - d e p e n d e n t       components                  ICl.2D f o r t i m e - d e p e n d e n t a n d Icl_21 f o r
c o m p o n e n t Ic]_2 was a C1 c u r r e n t t h a t r e q u i r e d Ca in-                      t i m e - i n d e p e n d e n t c o m p o n e n t s . As d i s c u s s e d below, we
q u x to b e activated.                                                                            b e l i e v e t h a t Icl_z~is actually IcH b e c a u s e it e x h i b i t s p r o p -
     Icz.2 consists of two components. T h e c u r r e n t t h a t we                              erties we w o u l d p r e d i c t f r o m t h e c h a r a c t e r i z a t i o n o f Icl-v
have t e r m e d Icl_2 c o n s i s t e d o f two c o m p o n e n t s : a time-                         Instantaneous IV relationship. T h e steady-state IV curve
independent component and a time-dependent com-                                                    in Fig. 6 s h o w e d t h a t ICI_2D i n w a r d l y rectified. T o d e t e r -
p o n e n t . T h e c u r r e n t - v o l t a g e r e l a t i o n s h i p s o f t h e time-        m i n e t h e cause o f this r e c t i f i c a t i o n , we m e a s u r e d t h e
dependent and time-independent components are                                                      i n s t a n t a n e o u s IV r e l a t i o n s h i p a n d t h e activation curve
p l o t t e d s e p a r a t e l y in Fig. 6. T h e t i m e - i n d e p e n d e n t                              2
                                                                                                   for ICI_ u s i n g tail c u r r e n t analysis. T h e i n s t a n t a n e o u s
c o m p o n e n t was m e a s u r e d as t h e c u r r e n t at 1,020 ms,                          c u r r e n t - v o l t a g e r e l a t i o n s h i p s were d e t e r m i n e d b e f o r e
a n d t h e t i m e - d e p e n d e n t c o m p o n e n t was m e a s u r e d as                    (Fig. 7 A) a n d ,'-45 m i n a f t e r (Fig. 7 B) i n j e c t i n g IP3. I n

                                              163           HARTZELL
this e x p e r i m e n t , the m e m b r a n e was h y p e r p o l a r i z e d to   K4-BAPTA followed ~ 5 m i n later by an injection o f 23
- 1 4 0 mV to activate Icl_2 a n d t h e n repolarized to differ-                   nl o f 10 m M IP 3. In oocytes that h a d b e e n injected with
e n t test potentials. D u r i n g the test pulse, an outward                       BAPTA, injection o f IP~ failed to i n d u c e any currents
c u r r e n t d e v e l o p e d with time. This c u r r e n t will be de-           (Fig. 10 A), whereas in c o n t r o l oocytes, IP~ injection
scribed in m o r e detail in Fig. 11, b u t in the p r e s e n t ex-                evoked b o t h Icl-i a n d Icl.2 in 95% o f the oocytes.
p e r i m e n t the instantaneous c u r r e n t was m e a s u r e d 4 ms               We also e x a m i n e d the ability o f BAPTA to block Icw
after the test step. T h e instantaneous c u r r e n t at the test                  after it h a d already d e v e l o p e d in response to IP3 injec-
potential reflected the c u r r e n t t h r o u g h the c h a n n e l s             tion. Injection o f BAPTA 10 m i n after IP~ injection h a d
that were o p e n at - 1 4 0 mV, before any c h a n n e l s h a d                   fully activated Icl_2 invariably b l o c k e d the time-depen-
time to o p e n o r close. T h e basal current-voltage rela-                        d e n t c o m p o n e n t ICl-ZD (Fig. 10 B). In this example, the
tionship slightly outwardly rectified a n d reversed at
- 2 5 mV, suggesting the p r e s e n c e o f a small basal C1
current. After injection o f IP> the c u r r e n t increased at
least 10-fold (Fig. 7 D). T h e current-voltage relation-
ship strongly outwardly rectified a n d reversed at the C1                                                         8000.
                                                                                     A                                      <
equilibrium potential. T h e t i m e - d e p e n d e n t c o m p o -
n e n t o f Icl_2 was completely b l o c k e d by 1 m M Mn (Fig. 7
C). It was also b l o c k e d by 0.5 m M niflumic acid.
                                                                                                                   6000.
                                                                                                                                e-


                                                                                                                                         /t IC1"2
    In the e x p e r i m e n t o f Fig. 7, we did n o t differentiate
between Io-2D a n d Io-2i T o d e t e r m i n e the i n s t a n t a n e o u s                                      4000.
IV relationship o f ICl.2D the IV curve o f the time inde-




                                                                                                                                                            Downloaded from www.jgp.org on February 20, 2005
p e n d e n t c o m p o n e n t (Fig. 6 A, squares) was subtracted
f r o m the IV curve o f the total Icl.z c u r r e n t (Fig. 7 D,                                                   2000

squares). Fig. 8 A shows these curves, a n d Fig. 8 B shows                            Voltage (mV)
                                                                                     -15o       -loo        -50
                                                                                                                     )
                                                                                                                   ~=/
                                                                                                                            L                   ICl.21
the subtracted IV relationship. T h e IV relationship o f
Io.z strongly outwardly rectified. This s e e m e d paradoxi-
cal, because the steady-state IV inwardly rectified. T o
resolve this p a r a d o x , we d e t e r m i n e d its activation                       ~   2     0    0    0
Curve.
   Activation curve. T h e activation curve for Io_2 is shown
in Fig. 9. T h e activation curve for Icl_2 was d e t e r m i n e d                                                 6000.       ~"
                                                                                                                                r.
by h y p e r p o l a r i z i n g to different pre-potentials to acti-
vate Icl_2 a n d t h e n repolarizing to + 2 0 mV to m e a s u r e                    B                                                         ICl-2D
the instantaneous o u t w a r d c u r r e n t t h r o u g h the chan-
nels that were o p e n at the e n d o f the prepulse. Fig. 9 A
shows a typical activation protocol, a n d Fig. 9 B shows
the average activation curve for five oocytes. Icl_9 b e g a n
to activate at potentials negative to - 3 0 mV a n d exhib-
ited a half-activation potential o f - 1 0 5 mV. At poten-
tials negative to - 1 5 0 mV, there was typically a de-                             Voltage( m V )
                                                                                                                    iiii
                                                                                                                      )     f        /
crease in Icl_ relative to the c u r r e n t at - 1 4 0 mV. T h e
                    2
activation curve in Fig. 9 B shows that the steady-state
                                                                                     -,s,0 -1~0 . -5~ .~]                            . s~ .
IV curve in Fig. 8 inwardly rectified because the chan-
nel did n o t activate at potentials positive to - 3 0 mY.
Different batches o f oocytes exhibited s o m e variability
in the half-activation potential ( + 10 mY). T h e reasons
for this variability have n o t b e e n investigated further.
                                                                                         ,,.i i000t
                                                                                    FIGURE 8. Instantaneous IV relationship for the time-indepen-
                                                                                                                                    2.
                                                                                    dent and time-dependent components of IcL_ To determine the
Icl-i and Icl2 Are Ca Activated                                                     instantaneous IV relationship of Icl.2Dthe IV curve of the time inde-
                                                                                    pendent component (Fig. 6 A, open squares) was subtracted from
We hypothesize that IcH is a Ca-activated C1 c u r r e n t                          the IV curve of the total Icy2 current (Fig. 7 D, open squares'). (A)
that is i n d u c e d by Ca released f r o m intracellular stores                   Time-independent component of Icl_2(open ,squares) from Fig. 6 A
                                                                                    and the instantaneous 1V relationship of the total Icl_ (solid2
a n d that Icl_2O is a Ca-activated C1 c u r r e n t that is in-
                                                                                    ,squares) from Fig. 8 A. (B) Subtraction of the two curves in A gives
d u c e d by Ca influx f r o m the extracellular space. As a                        the instantaneous IV relationship for the time-dependent compo-
test o f the hypothesis that these currents were Ca-acti-                                       ~
                                                                                    nent of Icl_ shows these curves, and B shows the subtracted IV rela-
vated, we first injected the oocyte with 46 nl o f 100 m M                          tionship.

                                       164       Different Cl Currents Activated by Ca Influx and Ca Stores
                                                                       +20mV
             -35mV
    A            --[                                                                                            B
                               -180mY
        8000
                                                                                                               1.0.


        6000
                                                                                                               0.8-
        4000
<                                                                                                       "m     0.6.
        2000                                                                                           (..)
"E
                                                                                                        t-     0.4.
                                                                                                        O

(..)         0

                                                                                                       I,.1_   0.2-
        -2000
                                                                                                               0.0.                                                                        B

        -4000              i        I         i                  i                  i
                                                                                                                                                                                                 i
                 o                ,oo                lo'oo             4o0                2o00                        -200                              -lao                                     o
                                                  Time (ms)                                                                                     Voltage (mY)
FIGURE 9. Activation curve o f Icl_2. T h e activation curve was d e t e r m i n e d by s t e p p i n g t h e m e m b r a n e to d i f f e r e n t p r e p o t e n t i a l s b e t w e e n




                                                                                                                                                                                                      Downloaded from www.jgp.org on February 20, 2005
- 160 a n d - 2 0 m V for 1-s f r o m a h o l d i n g potential o f - 3 5 m V a n d t h e n s t e p p i n g to a test potential o f + 2 0 m V for 1-s. (A) Traces s h o w i n g
t h e d e t e r m i n a t i o n o f t h e activation curve. (B) T h e c u r r e n t 4-ms after s t e p p i n g to t h e test potential was p l o t t e d as a f u n c t i o n o f t h e p r e p o -
tential. T h e c u r r e n t for e a c h oocyte was n o r m a l i z e d so that t h e c u r r e n t after t h e - 150 m V p r e p u l s e was set to 1.0. T h e data b e t w e e n - 1 5 0
a n d - 2 0 m V were fitted by t h e B o l t z m a n n e q u a t i o n : I = 1.05/[1 + e x p (v 10~)/15].



t i m e - i n d e p e n d e n t c o m p o n e n t Icl-2~was apparently not                           ship of IcH outwardly rectifies, whereas Ca influx will be
blocked, but this conclusion is complicated by the fact                                              greatest at hyperpolarized potentials, one would expect
that BAPTA alone often p r o d u c e d an increase in in-                                            steady-state IcH to be small at all potentials after stores
ward current that we have not fully characterized.                                                   are depleted. If this reasoning is correct, we should be
    To d e t e r m i n e the source of the Ca responsible for ac-                                    able to reactivate Icl-t after Ca stores are depleted by
tivation of ICI_2D, w e b a t h e d the cell in solution in which                                    first giving hyperpolarizing pulses to increase Ca influx
the CaC12 was replaced with BaC12 (Fig. 10, C and D).                                               followed by depolarizations to activate the CI channels.
U n d e r these conditions, injection of IP3 still activated                                        T o test this hypothesis, we p e r f o r m e d the test shown in
Icl4, but ICI_2D w a s not activated. T h e r e was an increase                                      Fig. 11. In this experiment, the oocyte was injected with
in inward current at - 1 2 0 mV that corresponded to Icl-zi.                                         IP 3 while stimulating with a voltage protocol similar to,
Similar results were observed when 1 mM Mn or La was                                                 but different from, the protocol used in Fig. 1. In this
a d d e d to the n o r m a l Ca-containing solution. Further-                                        protocol the m e m b r a n e was first stepped to - 1 2 0 mV
more, addition of Mn (Fig. 7 C) or removal of Ca                                                     and then to +20 inV. T h e rationale was that Ca enter-
blocked Icl_2O (see Fig. 13). These results are consistent                                           ing the cell when the driving force for Ca entry was
with the hypothesis that Ca influx is required for activa-                                           high at - 1 2 0 mV could activate IcH at +20 mV, a po-
tion of ICI_2D. O u r interpretation is that IP 3 first stimu-                                       tential which would o p e n the voltage gates for Ic1.1. Fig.
lates Ca release from internal stores which activates IcH.                                           11 A-Fshows the currents 40 s before and 40, 50, 100,
T h e "depletion" of Ca f r o m stores then activates capaci-                                        150, and 350 s after injecting IP3. At ~ 4 0 s after IP3 in-
tative Ca entry which is responsible for Icl_~Dactivation.                                          jection, IcH was activated exactly as described in Fig. 1.
                                                                                                     However, as IcH declined, it was replaced with an out-
                                                                                                    ward current exhibiting a different waveform that grew
                                                                                                     in amplitude in concert with the d e v e l o p m e n t of Icl_ 2.
Activation of It:t_1by Ca Influx
                                                                                                     T h e time course of d e v e l o p m e n t of these currents is
We believe that the kinetics of decline of IcH at +20                                                shown in Fig. 11 H, where the circles show the initial
mV in Fig. 1 reflects the time course of depletion of Ca                                             d e v e l o p m e n t of IcH m e a s u r e d at the end of the +20
stores. Fig. 1 B may give the additional impression that                                             mV pulse, the squares show the d e v e l o p m e n t of Icw at
IcH c a n n o t be activated in response to Ca influx, be-                                           the e n d of the - 1 2 0 mV pulse, and the triangles show
cause IcH declines (almost) to baseline within ~ 3 min                                               the current at 85 ms after the step to +20 mV. T h e in-
after IP 3 injection. However, this impression is incor-                                             activating outward current at +20 mV which peaks in
rect. Because the steady-state current voltage relation-                                             ~ 8 5 ms is referred to as 13.

                                             165         HARTZELL
                                                                                                                -35mV
                -35mV            -120mV                                    +20mV                                                                               -120mY
A                                                                                                   B
             lOOO


                                                                    BAPTA
    <           o                                                                                         -2000
    v
     t--                                              , , ,,              3
                                                                   BAPTA+IP
    "E


             -lOOO
                                                                                                          .-4000


             -2000           i         I          i                                                                  I             i    I           i    I          I       i            I   i

                     o               500                 1000        '     1500 '                                    0                 200              400     600                  800         10 0
                                                      Time (ms)                                                                                         Time (ms)


 C                                                                                                  D       -3Sm~                           +20mV

                                                                                                                                                                                -120mV
                                                                                                          2000




                                                                                                                                                                                                         Downloaded from www.jgp.org on February 20, 2005
                                           IP
             1500        Bariuma                ~ ~ = = = = = = ~
                                                                                                                               b


                                                                                                                                                                        Badum
             lOOO                                                                                   <
                                                                                                    r
                                                                                                           1000
                                                                                                    "E
                                                                                                                         a,r

        "~    soo                                                                                              0
                                                                                                                                                                    c
                                                                                                                                                                        f

                o                                                                                        -1000

                         6                      260                 460                  660              -2000 '                                            lOOO               lS'OO            2o'oo
                                                      Time (s)                                                                                          Time (ms)
FIGURE 10. Effect of Ba and BAPTA on the stimulation of IcH and Ic~_u. (A) The oocyte was first injected with 23 nl 50 mM K4.BAPTA.
This injection usually produced an increase in time-independent inward current. 5 rain later, 23 nl of 10 mM IP:~was injected. The super-
imposed traces shown were taken 5 min after BAPTA injection and 15 min after IP.~ injection. IP~ fails to stimulate IcH o r Icl_2. (B) Effect of
BAPTA injection o n Icl. 2. The cell was first injected with 4.6 nl 10 mM IP 3. The first trace shown was taken 15 min 'after IP:~injection. Subse-
quently, 23 nl of 50 mM BAPTA was injected. The BAPTA trace was taken 5 min after BAPTA injection. The time-dependent component
                                                                                                 ~.
of Icl.2 is completely blocked. (C) Effect of extracellular Ba on stimulation of IcH and Icl_ The CaC12 in the extracellular solution was re-
placed with 2 mM BaCI.~. At the time indicated, 4.6 nl 10 mM IP~ was injected while the cell was stimulated with a 1-s pulse to +20 mV fol-
lowed by a 1-s pulse to - ] 20 mV from a holding potential of - 3 5 mV. Open squares, current at the end of the + 20 mV pulse (IcH); open cir-
cles, current at the end of the - 120 mV pulse (Ic~_2). (D) Current traces from the same experiment as in C. The traces a (before IP:0, b (20 s
after IP:0, and c (400 s after IP3) correspond to the times indicated in C.


    The inactivating outward current at + 2 0 m V has character-                                    r e n t a n d t h e n s t e p p i n g to d i f f e r e n t p o t e n t i a l s f o r m e a -
istics oflcl+ W e h y p o t h e s i z e t h a t t h e i n a c t i v a t i n g o u t w a r d         s u r i n g t h e i n s t a n t a n e o u s c u r r e n t (Fig. 12 A). Fig. 12 A
c u r r e n t at + 2 0 m V 03) in this p r o t o c o l is ]Cl-1 b e c a u s e                       begins with the +80 mV pulse. The traces shown were
its i n s t a n t a n e o u s c u r r e n t - v o l t a g e r e l a t i o n s h i p r e s e m -     o b t a i n e d by s u b t r a c t i n g t h e c u r r e n t s b e f o r e i n j e c t i n g
b l e s Ic~.~ a n d b e c a u s e it a c t i v a t e s w i t h a t i m e c o n s t a n t            IP.~ f r o m t h o s e a f t e r i n j e c t i n g IP.~, t h u s t h e 1V r e l a t i o n -
v i r t u a l l y i d e n t i c a l to IcH. T h e s e f e a t u r e s a r e i l l u s t r a t e d   s h i p r e f l e c t s o n l y IP3-activated c u r r e n t . I d e n t i c a l results
in Fig. 12. T h e i n s t a n t a n e o u s IV r e l a t i o n s h i p o f t h e in-                w e r e also o b t a i n e d by s u b t r a c t i n g c u r r e n t s in t h e ab-
a c t i v a t i n g o u t w a r d c u r r e n t was d e t e r m i n e d in a n o o c y t e          s e n c e a n d p r e s e n c e o f C a in t h e b a t h . T h e i n s t a n t a -
p r e v i o u s l y i n j e c t e d w i t h IP.~ by s t e p p i n g t h e m e m b r a n e           n e o u s IV r e l a t i o n s h i p was n e a r l y l i n e a r (Fig. 12 B),
to - 120 m V f o r 1 s to p r o d u c e C a i n f l u x , f o l l o w e d by a                      w h i c h is v e r y s i m i l a r to t h e IV r e l a t i o n s h i p o f Icl. l (Fig. 3
s t e p to + 8 0 rnV f o r 50 ms to a c t i v a t e t h e o u t w a r d c u r -                     C). T h e s m a l l o u t w a r d r e c t i f i c a t i o n c o u l d p o s s i b l y b e

                                                 166           Different Cl Currents Activated by Ca Influx and Ca Stores
          -3,~v                            I         +~'nv         ]
                            -12OnlY
      4ooo      A                                                                B                                                               C
      2OOO




O
          0



                                      ,
                                           r



                                           i     ,     =     ,     i
                                                                                      t=40s              , ~ ' ~     f



                                                                                                                                                 ?
                                                                                                                                                  i
                                                                                                                                                      t=~0S




                                                                                                                                                          ,       i     ,
                                                                                                                                                                              f
                                                                                                                                                                              i       ,     =     ,      i
                                          1000       1500        2000             0           500        I000       1500      2000               0            500            1000         1500         2000

      4000

              D                                                                  E
      2000

              t = 1 0 0 s                                                         t= 150s                                                              t = 350s
         0
,3
     -2000
                             ~o           looo       1~o                                                                                         ()           .5~             1000        15~)         20~)
                                      Time (ms)                                                     Time (ms)                                                          l i m e (ms)




                                                                                                                                                                                                                     Downloaded from www.jgp.org on February 20, 2005
                                                                                                                   4000.

                  G                                                                             "3000
                                                                                                                   3000-
                                                                                                "2000
                                                                                                           v
                                                                                                                   2000~
                                                                                                - 1000
                                                                                                           c-

                                                                                                                   1000
                                                                                                "0
                                                                                                           O          0

                                                                                                                -1000-
         0                   500           1000             1500        400_ a ~ ~p ~n~o~~~se ,J
                                                                                                                              6         1()0          2()0            3(30        4()0           560          6()0
                                                                                                                                                              Time (s)
                                      Time (ms)
FIGURE 11. An inactivating time-dependent outward current (I:~) develops slowly after IP3 injection. The experimental design is similar to
that in Fig. 1, except that the voltage protocol was reversed: a 1-s pulse to - 120 mV was followed by a pulse to +20 mV from a holding po-
tential of - 35 InV. A-Fshow current traces before and at various times after IP 3 injection. Note the change with time of the kinetics of the
outward current at +20 mV. At t = 50s (C) the current does not inactivate. It then declines in amplitude (D) and is replaced by an inacti-
~ating outward current (F) that grows in amplitude concurrently with the development of IcI-2" ( G ) Another way of displaying the data of
A - F t o give a more dynamic view of the change in the outward current. 40 traces are stacked together in the z-axis starting with the first
trace at the back and ending with the trace at 400 s after IP3 injection at the front. The x-axis is the time of the 2-s voltage pulse and the
y-axis is the amplitude. (H) The time course of change in currents after IP3 injection. Open circles, I(-H measured as the current near the
end of the + 20 mV pulse (1,950 ms); open squares, Icl.2 measured as the current near the end of the - 120 mV pulse (900 ms); filled triangles,
peak of the transient outward current (I3), measured as the current at 1,085 ms.




d u e to c o n t a m i n a t i o n o f this c u r r e n t w i t h Icl_2 w h i c h                              The inactivating outward current activates with the same
m a y n o t c o m p l e t e l y d e a c t i v a t e d u r i n g t h e 50 m s p u l s e                      kinetics as Ict-r W i t h t h e v o l t a g e p u l s e to + 2 0 m V , t h e
to + 8 0 mV. W e h a v e n o t s u c c e e d e d in s e l e c t i v e l y b l o c k -                       i n a c t i v a t i n g o u t w a r d c u r r e n t a c t i v a t e d w i t h a s i n g l e ex-
i n g Icl. 1 o r Icl_2 so it h a s b e e n i m p o s s i b l e to e v a l u a t e t h e                     p o n e n t i a l h a v i n g ~" = 25 m s (Fig. 12 D), w h i c h is v e r y
c o n t a m i n a t i o n o f this o u t w a r d c u r r e n t w i t h t h e u n d e r -                    s i m i l a r to t h e "r o f t h e fast c o m p o n e n t o f t h e a c t i v a t i o n
l y i n g d e a c t i v a t i o n o f Icl.2. H o w e v e r , t h e IV c u r v e is                          o f IcH (Fig. 12 C). T h e d e c l i n e o f t h e c u r r e n t o c c u r r e d
m a r k e d l y d i f f e r e n t f r o m t h a t o f Icl-2, s u g g e s t i n g t h a t                    w i t h a "r = ~ 2 5 0 ms, w h i c h w e p r e s u m e is t h e t i m e c o n -
c o n t a m i n a t i o n is small.                                                                         stant of reduction of cytosolic Ca upon depolarization.

                                                     167           HARTZELL
                                                                                                                                                 FW,  URE 12. Instantaneous cur-
                                                                                                                                                 rent-voltage relationship and ki-
                                                                                                                                                 netics of I.~suggests it is probably
                                                                                                                                                 I(;H. (A) Instantaneous IV of [~.
 A                                                                                                                                               The oocyte was stepped to -120
                                                                                       B                         6000 (~"                        mV ff)r 1-s before the start of the
        -120mV                                  -120mV                                                                                           trace shown. The membrane was
                                                                                                                                                 then stepped to +80 mV for 50-
          4000                                                                                                   4000                            ms tollowed by steps to different
          3000 #~...~                                                                                                                            test potentials. (B) The ampli-
                                                                                                                 2000                            tude of the current 4 ins after
          2000              ~.~                                                                                                                  stepping to the test potential was
                                                                                       , Voltage. ImV).      , /.-
                                                                                                                                                 plotted vs. the test potential. The
          1000
                                                                                                                                  5JO    100     data are the averages of six oo-
                                                                                  -150         -100 S/~'~                                        cytes. The straight line is drawn
   L)        o

          -1000
          -2000
             1000          1~o              11~o               11~o
                                                                                                                .2oo.4001                        to indicate a linear IV relation-
                                                                                                                                                 ship for a C1 conductance. ((3
                                                                                                                                                 Kinetics of activation and deacti-
                                                                                                                                                 vation of I:~.The outward current
                                 Time (ms)                                                                                                       was activated by a pulse to +20
                                                                                                                                                 mV after a 1-s prepulse to -120
                                                                                                                                                 inV. The activation (0-100 ms)
                                                                                                                                                 was fitted to a single exponential
     C                                                                                                                                           with a -r = 25 ms. The deactiva-
                                                                               DO00
                                                                                                                                                 tion (200-800 ms) was fitted by a




                                                                                                                                                                                              Downloaded from www.jgp.org on February 20, 2005
          4000
                                                                                                                                                 single exponential with "r = 246
                                                                                 800
                                                                                                                                                 ms. The thin lines show the fits
          3OOO
                                                                                                                                                 and the noisy trace is the data.
                                                                                                                                                 (D) Kinetics of activation of It:H.
          2000                                                            t-                                                                     Icl-i was activated by IP:~injection
    ==
                                                                                 40o                                                             and voltage steps from - 3 5 to
   (~ 1000                                                                0                                                                                                   ~
                                                                                                                                                 +20 inV. The onset of Ic~_ is fit to
                                                                                 2OO                                                             the sum of two exponentials with
                                                                                                                                                 "r = 28 and 274 ms. The fit is the
                                                                                                                                                 dashed line, which is very diffi-
                  -6   ' 2~0      '   4~o   '    ~o      '   a~o      '           o      0         200           400        600                  cult to distinguish from the noisy
                                 Time (ms)                                                                Time (ms)                              data trace.


Effects of Extracellular Ca                                                                               t h e n r a n o u r s t a n d a r d voltage protocol: a 1-s pulse to
                                                                                                          - 1 2 0 mV followed by a 1-s pulse to + 2 0 m V f r o m a
"Capacitative Ca entry" is classically d e m o n s t r a t e d by
                                                                                                          h o l d i n g p o t e n t i a l of - 3 5 mV. Fig. 13 B - E shows the
d e p l e t i n g i n t r a c e l l u l a r Ca stores (with IP~ o r thapsigar-
                                                                                                          c u r r e n t traces b e f o r e a n d at d i f f e r e n t times after re-
gin) i n the a b s e n c e o f e x t r a c e l l u l a r Ca a n d t h e n mea-
                                                                                                          a d d i t i o n o f 2 m M Ca. At 6 s after a d d i t i o n o f Ca, a large
s u r i n g the c u r r e n t that is s t i m u l a t e d by a d d i t i o n o f ex-
                                                                                                          Icl.2O c u r r e n t was r e c o r d e d at - 1 2 0 m V followed by I(:~.1
tracellular Ca. If Icl.2 is activated by Ca influx, we w o u l d
                                                                                                          at + 2 0 mV. Both Icv2D a n d IcH i n c r e a s e d t r a n s i e n t l y af-
e x p e c t it to be activated u p o n r e - a d d i t i o n of Ca to the
                                                                                                          ter a d d i t i o n o f Ca a n d t h e n d e c l i n e d to a lower p l a t e a u
b a t h after i n j e c t i o n o f IP~ i n Ca-free solutions. Fig. 13
                                                                                                          that r e m a i n e d relatively c o n s t a n t d u r i n g the r e m a i n d e r
shows the effect o f switching f r o m 0-Ca R i n g e r to 2 m M
                                                                                                          of the r e c o r d i n g . T h e time course of c h a n g e o f the cur-
Ca R i n g e r a p p r o x i m a t e l y 10 m i n after i n j e c t i o n o f [P~.
                                                                                                          r e n t s (Fig. 13 F) closely a p p r o x i m a t e d the time course
T h e cell was voltage c l a m p e d at - 6 0 mV, a n d the hold-
                                                                                                          of d e c l i n e of the h o l d i n g c u r r e n t at - 6 0 m V (Fig. 13
i n g c u r r e n t was m e a s u r e d . Switching f r o m 0 to 2 m M
                                                                                                          A). T h e d e c l i n e i n c u r r e n t is p r e s u m a b l y d u e to partial
Ca i n oocytes that h a d n o t b e e n i n j e c t e d with IP 3 usu-
                                                                                                          refilling of the Ca store by Ca influx.
ally p r o d u c e d n o c h a n g e i n the h o l d i n g c u r r e n t at - 6 0
mV. However, as s h o w n i n Fig. 13, w h e n the oocyte h a d
                                                                                                          Putative Store-operated Ca Current in Oocytes
previously b e e n i n j e c t e d with IP3, a d d i t i o n o f Ca pro-
d u c e d a r a p i d i n c r e a s e in i n w a r d c u r r e n t . T h e c u r r e n t                  O u r i n t e r p r e t a t i o n of the data above p r e s u m e s that
r e a c h e d a p e a k o f - 1 , 0 0 0 n A in several s e c o n d s a n d                                t h e r e is a s t o r e - o p e r a t e d Ca c u r r e n t in oocytes. We have
t h e n rapidly d e c l i n e d to a p l a t e a u level o f - 2 5 0 hA,                                  a t t e m p t e d to m e a s u r e this c u r r e n t directly as s h o w n in
w h e r e it r e m a i n e d at least 10 m i n . T o d e t e r m i n e which                              Fig. 14. I n this e x p e r i m e n t , we first i n j e c t e d IP 3 to re-
c u r r e n t s were r e s p o n s i b l e for this i n w a r d c u r r e n t at                          lease Ca f r o m i n t r a c e l l u l a r pools a n d t h e n i n j e c t e d
- 6 0 mV, we r e t u r n e d the oocyte to 0-Ca R i n g e r a n d                                         BAPTA to i n h i b i t Ca-activated C1 currents. T h e cell was

                                            168              DifferentC1 Currents Activated by Ca b~ux and Ca Stores
        A
                        2raM Ca          Time (sec)
                                                                                          -35m

                                                                                       3000    ]B             -120rnV
                                                                                                                             II         §



                                  25       50          75             100              2000                  0-Ca
               0                  I        t               I            I


                                                                    9~21o64 ~          lOOO

            -250                                                                           o


                                                                                       -1000

            -500
 r--                                                                                   -2000
                                                      =_

 t-
                                                                                       4000
 ,,~        -750
r                                                                                                C           t = 6s                                        FIGURE 13. Effect of changing
                                                                                                                                                           extracellular Ca on oocyte cur-
                                                                                       2000
                                                                                                                                                           rents. The oocyte was injected
        -1000
                                                                               E                                                                           with IP3 with the oocyte bathed
                                                                               B           o                                                               in normal Ringer about 10 min
                                                                                                                                                           before the beginning of the re-
                                                                                                                                                           cordings shown. After the devel-
                                                                                       -2000                                                               opment of Io_2 the oocyte u~as
                                                                                                                                                           shifted into 0 Ca Ringer for ~ 2
                                                                                                                                                           rain. (A) Effect of re-addition of
                                                                                       3000                                                                2 mM Ca to an oocyte bathed in




                                                                                                                                                                                                      Downloaded from www.jgp.org on February 20, 2005
                                                                                                     D         , = 22s                                     0-Ca Ringer after IPs injection9
                                                                                       2000
                                                                                                                                                           The membrane potential was
    F       4000-                                                                                                                                          held at - 6 0 mV. The same oo-
                                                                      CI-21            10001
                                                                                                                                                           cyte was then shifted back to 0-Ca
            3000                                                                           0                                                               Ringer for ~ 2 rain and the stan-
                                                                                                                                                           dard -120 to +20 mV voltage
                                                                                       -1000                                                               protocol (Fig. 11) was applied
<           2000-                                                                                                                                          once every 3 s9 (B) A sample cur-
E
                                                                                       -2000                                                               rent trace in response to the
            1000-                                                                      3000                                                                -120 to 20 mV protocol in 0-Ca
                                                                                                     E                                                     Ringer. (C~s Current traces re-
                                                                                       2000                                                                corded 6, 22, and 50 s after addi-
               0-                                                                                            t = 50s
                                                                                                                                                           tion of 2 mM Ca to the bath. (F)
                                                                                       1000                                                                Plot of the change in currents
        -1000,                                                                     E
                                                                                                                                                           with time after adding 2 mM Ca.
                                                                                           0                                                               Filled squares, time-dependent
                                                                                                 =

                                                                                                                                                           component of Icl_2; open circles,
                                                                    96321064           -1000
        -2000                                                                                                                                              time-independent component
                    0             2'o          go              ~o                                                                                          of Icl_Z; open triangles, peak tran-
                                                                                       -2000             ,       ,      ,    ,      .       i   .    ,

                                        Time (s)                                               0               500          1000        1500        2000   sient outward I~ current at +20
                                                                                                                        Time (ms)                          mV (measured at 1,085 ms).




s t i m u l a t e d with 2-s d u r a t i o n r a m p pulses f r o m - 1 6 0 to                                served. F u r t h e r m o r e , this c u r r e n t activates i n a voltage
50 inV. U n d e r these c o n d i t i o n s , shifting f r o m 0-Ca to                                        r a n g e that e x p l a i n s the activation o f [c].2
10 m M Ca p r o d u c e d a shift i n the r a m p c u r r e n t (Fig.
14A). T h e d i f f e r e n c e in the r a m p c u r r e n t s i n the ab-
s e n c e a n d p r e s e n c e of Ca (Fig. 14 B) r e s e m b l e s the                                       DISCUSSION

s t o r e - o p e r a t e d Ca c u r r e n t Icrac i n T-lymphocytes a n d
                                                                                                              Conclusions
mast cells ( H o t h a n d P e n n e r , 1993; Zweifach a n d
Lewis, 1993; P r e m a c k et al., 1994). We c a n n o t rule o u t                                           Xenopus oocytes exhibit two different Ca-activated Cl cur-
the possibility that the d i f f e r e n c e c u r r e n t is residual Ca-                                    rents. I n these studies we have s h o w n that i n j e c t i o n o f
activated C1 c u r r e n t n o t b l o c k e d by BAPTA, b u t the fact                                       IP~ i n t o Xenopus oocytes activates two d i f f e r e n t Ca-acti-
t h a t t h e r e is n o o u t w a r d c u r r e n t at + 5 0 m V (Fig. 14 B)                                vated C1 c u r r e n t s . IcH is s t i m u l a t e d rapidly (within 5 s
a r g u e s a g a i n s t this possibility. T h e c u r r e n t is b l o c k e d by                          after IP~ i n j e c t i o n ) , exhibits t i m e - d e p e n d e n t activation
La, a n d its a m p l i t u d e is r e l a t e d to the e x t r a c e l l u l a r                            u p o n d e p o l a r i z a t i o n , a l i n e a r i n s t a n t a n e o u s IV rela-
[Ca] a n d r e q u i r e s p r i o r s t i m u l a t i o n with IP~ to be ob-                                t i o n s h i p with a reversal p o t e n t i a l n e a r Ec], a n d a curvi-

                                               169    HARTZELL
         A                                             ,00{                         Icl-1 is stimulated both by Ca released from stores and by Ca
                                                                                 influx. We conclude that Ic14 is stimulated by Ca re-
                                                                                 leased f r o m pools for the following reasons: (a) Intra-
                                                                                 cellular injection of IP~ rapidly stimulates Icu, and the
                                                       200.L                     stimulation is blocked by intracellular injection of
                                                                                 BAPTA. This suggests that Icu is stimulated by Ca and
             -150         -100           -50 , - - S                        50   not by IP 3 directly. (b) Injection of IP 3 stimulates IcH
               I    ,          I   ,      I                             ,    I   even when Ba or Mn replace extracellular Ca, suggest-
Voltage (mY)                                                                     ing that the source of stimulating Ca is not Ca influx
                                                               <
                                                               v
                                                                   e"            but rather release of Ca f r o m stores. In our analysis of
                                                     -200      E                 the properties of Icu in Figs. 2-4, we presume that
              o
                                                                                 [Ca]i at short times after IP.~ injection is relatively inde-
                        lOCa                                   O                 p e n d e n t of m e m b r a n e potential because Ca influx
                                                                                 through store-operated channels has not yet been acti-
                                                     -400                        vated. Furthermore, the voltage steps used to deter-
                                                                                 mine the activation curve were very brief and currents
                                                                                 were measured 4 ms after the voltage steps. O n e would
                                                                                 not expect cytosolic Ca to change significantly on this
     g                                                 40~
                                                                                 time scale. Thus, the curvilinear activation curve of IcH

         ,51 ,01                                       2ot                       reflects voltage-dependent gating of the channel and
                                                                                 not the level of cytosolic free Ca.

                                   ~~




                                                                                                                                                                  Downloaded from www.jgp.org on February 20, 2005
                                                                                    In addition to actitvation by store Ca, Icu can be stim-
 Voltage (my)                                                                    ulated by Ca influx after the stores have b e e n depleted,
                                                                                 provided that Ca influx is a u g m e n t e d by increasing the
                                                                                 driving force for Ca entry by hyperpolarization im-
                                                                                 mediately before voltage-dependent activation of I(:14
                                                                                 by a depolarization. T h e slowly activating current u p o n
                                                                                 stepping to +20 mV f r o m - 1 4 0 mV after IP3 injection
                                                                                 is Icl-l. T h e r e are several reasons why we believe that the

             /'                                     -,~176 i
                                                    -120
                                                                                 inactivating outward current that develops with time al-
                                                                                 ter IP 3 injection u p o n depolarization to +20 mV f r o m
                                                                                 - 1 4 0 mV (Fig. 11) is IcH: (a) T h e instantaneous cur-
FIGURE 14. Putative store-operated Ca current in oocytes. The                    rent-voltage relationships of the inactivating outward
oocyte was injected with IP3 "~ min and with BAPTA "-d0 min                      current at +20 mV and IcH are both linear. (b) Both
before the records shown here. The oocyte was placed in 0-Ca                     currents activate with the same kinetics (~ = 25 ins). (c)
Ringer for 2 min, and the current recorded in response to a 2-s du-              T h e activation cmwe for Icu predicts that m o r e Icl_1 will
ration ramp pulse from -160 to +50 mV. The oocyte was shifted                    activate with depolarizations to any potential positive to
to 10 mM Ca and the current in response to the same ramp was re-
corded. The difference between these two currents (bottom) is the                - 1 4 0 mV (Fig. 4). In fact, Fig. 7 B shows that u p o n
presumed store-operated Ca influx.                                               stepping f r o m - 1 4 0 mV to various potentials, inward
                                                                                 currents are activated negative to Ec~ whereas outward
                                                                                 currents are activated positive to Eel. This interpreta-
                                                                                 tion differs f r o m that of Yao and Parker (1993) (see
                                                                                 below).
linear activation curve with an a p p r o x i m a t e half-maxi-                                                                                  2
                                                                                    T h e t i m e - i n d e p e n d e n t c o m p o n e n t of Ic~_ is probably
mal activation voltage of >200 mV. Ic~_ a p p e a r e d to be
                                               2                                 Icl.1 for the following reasons: (a) O n e would expect an
c o m p o s e d of two components. T h e t i m e - d e p e n d e n t             inward current through Icl-i channels at potentials be-
c o m p o n e n t ICI_2Dis stimulated slowly after IP3 injection                 tween Eel and - 1 5 0 mV based on its activation curve
(half-maximal stimulation occurs ~ 3 min after injec-                            (Fig. 4). (b) Both Ic1-1 and Icl-2i have a linear current-
tion). ICI_2Dhas a strongly outwardly rectifying instanta-                       voltage relationship (Fig. 3 C and 8 A). (c) It.vl and I(:l_,21
neous IV relationship with a reversal potential near Ec~                         exhibit identical time courses in Ca-jump experiments
and is activated by hyperpolarization with a half-maxi-                          (Figs. 13 and 14). The only e x p e r i m e n t in possible con-
mal activation voltage of - 1 0 5 mV. T h e time-indepen-                        flict with this interpretation is that Ic1_2iwas apparently
dent c o m p o n e n t Ic1.21 has a linear current-voltage rela-                 not blocked by internal BAPTA (Fig. 10 B). However,
tionship and a reversal potential near Ecl and, as dis-                          this e x p e r i m e n t may be misleading because injection of
cussed below, is actually Icu.                                                   BAPTA itself before IP3 produces an increase in inward

                                   170        Different Cl Currents Activated by Ca Influx and Ca Stores
current. Thus, the a p p a r e n t absence of effect of
BAPTA on the t i m e - i n d e p e n d e n t inward current may
be due to two opposite effects. Icl.2i is completely
blocked by 0-Ca extracellular solution (Fig. 13).
    ICl-eD is stimulated by Ca influx through store-operated Ca
channels. We hypothesize that ICI_2D is preferentially ac-
tivated by Ca influx t h r o u g h store-operated Ca chan-
nels for the following reasons: (a) Icl-2Drequires Ca for
activation, as it is blocked by intracellular injection of
BAPTA. (b) ICI.2Drequires Ca influx for activation, be-
cause it is blocked by addition of Ba or Mn to the extra-
cellular solution or removal of extracellular Ca. (c) The
time course of stimulation of Icl-2Dcorresponds with the
time course of emptying intracellular Ca stores as as-
sessed by the decline of Ici.1. (d) T h e time course of ac-
tivation of Icl. 2 u p o n stepping f r o m - 3 5 to - 1 2 0 mV
reflects the time course of accumulation of Ca in the cy-
tosol as m e a s u r e d by the amplitude of IcH on stepping
to + 20 mV at different times during the hyperpolariz-
ing step (data not shown).
    The Ca concentration~jump e x p e r i m e n t of Fig. 13




                                                                                                                                             Downloaded from www.jgp.org on February 20, 2005
provides additional insight into the regulation of I~l_2D.
W h e n the oocyte is shifted f r o m 0-Ca to 2 mM Ca after
IP 3 injection, the first hyperpolarization produces a
large Icl.2. This declines rapidly to a steady-state level
that is less than half the amplitude of the initial cur-
rent. We p r e s u m e that this decline reflects refilling of
the Ca stores over this ~15-s period and a subsequent
decrease in Ca influx. W h e n the oocyte is shifted to 10
mM Ca (data not shown), the initial Ic~_zis extremely
large, but it declines even m o r e rapidly than with 2 mM
Ca and quickly disappears. However, even though Icl. 2
has disappeared, IcH remains very large. T h e r e are sev-
eral possible explanations of this result. O n e possibility
is that the Ca influx refills the Ca stores, and the store-
o p e r a t e d Ca channels subsequently close completely,
but IcH remains elevated because of oscillatory Ca re-
lease f r o m stores.
    Store-operated Ca current. The activation of Icl.,~ by hy-
perpolarizing steps (Fig. 9) in principle could be ex-
plained either by strictly voltage-dependent gating of              FIGURE 15. Model of regulation of C1 channels in Xenopus oo-
the C1 channels or could result indirectly f r o m the volt-        cytes. (Top) When the oocyte is injected with IP3, Ca is mobilized
a g e - d e p e n d e n t influx and accumulation of Ca. The lat-   from stores and I~:l_lis induced. The steady-state current-voltage re-
                                                                    lationship of Ic:His shown above. (Bottom) Alter some minutes, de-
ter possibility seems m o r e likely because the putative           pletion of the store triggers opening of store-operated Ca channels
store-operated Ca current (Fig. 14) predicts the activa-            (possibly via a calcium influx factor, CIF). Ca influx then induces
tion of Ic912. T h a t is, the store-operated current activates     both Ic~_land Icl_2, The steady-state current voltage relationships
significantly only at potentials negative to - 5 0 mV, as           are shown above to indicate that the currents that flow depend
does Icl. z. This current-voltage relationship for the              upon potential. Ca influx may also serve to replenish the pool.
store-operated Ca influx explains why the inactivating
outward ICH current is present only when the m e m -
b r a n e is hyperpolarized to potentials negative to - 5 0         m e n t that this current is a store-operated current. The
mV before the depolarizing step (Fig. 9 A). T h e obser-            o t h e r arguments are that this current is d e p e n d e n t on
vation that the voltage range for activation of outward             extracellular Ca, is blocked by La, and is present when
Ic1.1 parallels the activation range for the putative store-        all Ca-activated C1 currents m e a s u r e d by o u r conven-
o p e r a t e d current (Fig. 14) provides an additional argu-      tional protocols are blocked by intracellular BAPTA.

                               171     HARTZELL
   Model of regulation of Ca-activated Cl channels in Xenopus        could not be resolved f r o m the capacitative transient)
oocytes. O u r m o d e l for regulation of C1 channels by Ca         with the resulting Ca influx activating C1 channels. In
is shown in Fig. 15. Injection of IP3 stimulates release of          fact, however, the contribution of Ca influx via voltage-
Ca f r o m stores, which rapidly stimulates Ic~_l channels           gated channels is not i m p o r t a n t because the envelope-
( u p p e r panel). As the stores b e c o m e depleted, store-       of-tails test (Hodgkin and Huxley, 1952) shows that the
operated Ca channels are activated which can stimulate               tail currents precisely predict the time course of Ic~_>
both IcH and Icl_ d e p e n d i n g on potential (lower
                         2                                           which would not be true if a voltage-gated Ca influx
panel). It should be n o t e d that because we have not in-          were contributing to currents r e c o r d e d during the tail.
hibited the Ca-ATPase, Ca uptake into the SR has not                 F u r t h e r m o r e , neither 100 ~M nickel n o r 10 txM nife-
b e e n inhibited. Thus, activation of IcH at long times af-         dipine, which block the e n d o g e n o u s T-like and L-like
ter IP:~ injection could actually be due to Ca entering              Ca currents in tile oocyte (Barish, 1983; Dascal et al.,
the cell via influx subsequently entering Ca stores and              1986; Dascal, 1987), had any effect on the shape of the
subsequently activating IcH.                                         Icl-2 1V relationship, although they both decreased the
   At present we have no explanation why store Ca is                 amplitude of Icl_2 :'-50%.
not capable of activating It>). It seems unlikely that the
explanation is that IcH and Icl.~) have different sensitivi-         Relationship to Other Studies
ties to Ca. Approximately the same a m o u n t of IcH is ac-         Since the early 1980's, a great deal has b e e n learned
tivated by store Ca and by Ca influx (Fig. 11). If we as-            a b o u t Ca signaling and C1 channels in Xenopus oocytes
sume that the amplitude of Icl_1 is a relative measure of            largely as the result of the pioneering work of R. Miledi
the a m o u n t of Ca n e a r these channels, this suggests          and I. Parker, Y. Lass and N. Dascal, and M. Berridge
that approximately the same a m o u n t of Ca is present at




                                                                                                                                              Downloaded from www.jgp.org on February 20, 2005
                                                                     and R.F. h'vine and their colleagues. The ionic currents
the m e m b r a n e u n d e r these two conditions. Thus, it         that we have described here have b e e n observed by
seems that spatial and temporal aspects of Ca concen-                these investigators previously. However, the present
tration must be i m p o r t a n t (Lechleiter and Clapham,           study adds to the earlier ones by characterizing the bio-
1992).                                                               physical properties of these channels in m o r e detail to
                                                                     show that there are clearly two different kinds of Ca-
Voltage gated Ca Current
                                                                     activated C1 channels that are regulated differently.
T h e above conclusions could be c o m p r o m i s e d if Ca in-        IP~ activates C1 currents with several components. In read-
flux through voltage-gated Ca channels was partly re-                ing the literature on Xenopus oocytes, one can divide CI
sponsible for activation of these C1 currents. Xenopus               current responses to agents that mobilize store Ca into
oocytes have b e e n shown to have e n d o g e n o u s voltage-      three different groups based on their time courses and
gated Ca channels (Dascal, 1987) that might be ex-                   d e p e n d e n c e on extracellular Ca. The first response is a
pected to contribute Ca influx to activate C1 currents.              transient current that activates and inactivates in :"2
Such a C1 c u r r e n t (Icl(ca)) activated by Ca influx through     min and is not d e p e n d e n t on extracellular Ca. This
w)hage-gated channels has b e e n described by Barish                current is very similar to IcH and is likely to be caused
(1983) and Miledi (1982). However, there are a num-                  by release of Ca f r o m stores by IP3. The second compo-
ber of reasons why Ca influx via voltage-gated channels              n e n t is also i n d e p e n d e n t of extracellular Ca, but devel-
is unlikely to contribute significantly to the currents de-          ops slowly after release of store Ca. This current may be
scribed here. (a) Icl(c~) described by Barish (1983) is at           activated by a slower mobilization of store Ca u n d e r
least 10-times smaller than the currents we have de-                 certain conditions and may also be mediated by IcH.
scribed here with physiological concentrations of exter-             The third c o m p o n e n t is identical to I(,1_2 in that it is acti-
nal Ca. (b) T h e IV relationship for Icl(c~) is A-shaped            vated at hyperpolarizing potentials and requires extra-
with a peak n e a r 0 mV, reflecting the IV relationship of          cellular Ca. Despite the fact that it is possible to identify
the voltage-gated Ca channels (Barish, 1983). Neither                these three kinds of responses based on time course
IcH nor Icl_~ exhibit an obvious c o m p o n e n t with this         and sensitivity to Ca,,, it is difficult to c o m p a r e results of
shape IV relationship. (c) In the experiments on IcyI in             different investigators unambiguously, because the cur-
Figs. 2-4, IcH did not require extracellular Ca. Further-            rent responses in the various studies were often not
more, the holding potential was - 3 5 mV which would                 characterized in sufficient detail either biophysically or
largely inactivate the voltage-gated Ca channels (Dascal             pharmacologically. For example, current voltage rela-
et al., 1986). Thus, the properties of Icl_1 activated by            tionships of currents that one might expect to be the
store Ca would not be convoluted by any contribution                 same based on their time course and Ca,, sensitivity are
from I(:l((:~,). (d) In principle, Ca influx via voltage-gated       often different.
Ca channels could possibly c o n f o u n d the tail analysis of         Most publications have focussed on only one or two
I(>> if the test depolarizations after the - 1 2 0 mV steps          of these components, but all three c o m p o n e n t s were
(Fig. 7) were to activate voltage-gated Ca channels (that            observed in the same oocytes by Snyder et al. (1988),

                              172     Diffi,ren! C1(h,rrents Activated by Ca Influx and Ca Stores
who d e m o n s t r a t e d that IP3 injection into the oocyte in-      a g o n i s t . Tin is a CI current with a steady-state current-
d u c e d an inward current at - 5 0 mV holding potential               voltage relationship that resembles Icl.2. The current is
having three c o m p o n e n t s . O n e c o m p o n e n t called Ila   blocked by Mn-containing or 0-Ca extracellular solu-
was i n d e p e n d e n t of Ca influx and activated and inacti-        tions and is blocked by EGTA injection into the oocyte.
vated in :"2 rain. Ilb w a s also i n d e p e n d e n t of Ca influx,   This current is also activated in native oocytes by injec-
but activated m u c h m o r e slowly and lasted "-q0 min or             tion of IP 3. Petersen and Berridge (1994) have shown
m o r e . I 2 was d e p e n d e n t on Ca influx and activated          that LPA (via a PLC-coupled receptor), thapsigargin,
slowly. It would seem that the fast Ca-influx indepen-                  a n d IP.~ activate currents that resemble IcH and Icl_2
d e n t c o m p o n e n t corresponds to o u r Icl. 1 a n d the slow    with regard to their differing d e p e n d e n c e on Cao and
Ca-influx d e p e n d e n t c o m p o n e n t corresponds to o u r      time course. Indeed, Petersen and Berridge (1994)
Icl_z but the biophysical properties of these currents                  have presented data similar to o u r Fig. 13, but they
were not d e t e r m i n e d (Snyder et al., 1988). O t h e r stud-     seem to interpret the data in terms of a single C1 con-
ies usually describe two components, but the dependence                 ductance rather than multiple conductances.
o f the c o m p o n e n t s on Ca influx is rather variable.                Stimulation o f the serotonin (5-HT) (1 C) receptor in
    In some studies, IP.~ injection or activation of recep-             oocytes also produced an initial rapid oscillatory current
tors that activate phospholipase C induced two Ca-                      followed by a p r o n o u n c e d secondary current (Parekh
d e p e n d e n t C1 currents that did not require Ca influx.           et al., 1993), but separation of the two c o m p o n e n t s into
In response to IP~ injection, a transient current devel-                Cao-dependent and i n d e p e n d e n t c o m p o n e n t s was not
o p e d rapidly u p o n IP3 injection and this was often fol-           as clear. The initial c o m p o n e n t was reduced :-,35% by
lowed by a m o r e slowly developing c o m p o n e n t that was         0-Ca and thus may be partially d e p e n d e n t on Ca influx.
frequently oscillatory in nature ( O r o n et al., 1985;                T h e secondary c o m p o n e n t was blocked by 100 b~M Cd




                                                                                                                                                Downloaded from www.jgp.org on February 20, 2005
Parker and Miledi, 1986; Berridge, 1988; Parker and                     and was sensitive to the concentration of extracellular
Ivorra, 1991). T h e amplitude of the second c o m p o n e n t          Ca, suggesting that this c o m p o n e n t required Ca influx.
s e e m e d to be quite variable in amplitude f r o m oocyte            In these same studies, injection of IP 3 p r o d u c e d vari-
to oocyte (Parker and Ivorra, 1991). Activation of mus-                 able responses: either an oscillatory current or a rapid
carinic receptors also activated two consecutive inward                 current followed by a sustained secondary one. T h e sus-
C1 currents at - 6 0 mV (D~ and D~ responses) that were                 tained current was only partly blocked by Cd, suggest-
m i m i c k e d by IP3 injection and were i n d e p e n d e n t of      ing that there may be overlapping c o m p o n e n t s that are
Cao ( O r o n et al., 1985; Gillo et al., 1987). T h e r e does         d e p e n d e n t on Ca influx and Ca release f r o m stores un-
not seem to be a g r e e m e n t a b o u t the shape of the IV re-      der these conditions.
lationships of the currents induced by IPa or receptor                      T h e r e are several possible explanations why some in-
activation. T h e currents have b e e n described as both               vestigators observe an I t : l i k e current a n d others do
weakly outwardly rectifying (Dascal et al., 1984; O r o n et            not. O n e explanation is that the ability to observe Ic~_          2
al., 1985) and very strongly outwardly rectifying (Miledi               depends on the voltage protocol used. At holding po-
et al., 1989). A strongly rectifying Ca-activated CI cur-               tentials positive to - 6 0 mV, activation of Icl-2 is small
rent is also induced by injection of Ca into oocytes                    (Fig. 9). F u r t h e r m o r e , activation of Icl-2 requires larger
(Miledi and Parker, 1984).                                              amounts of IP~ than activation of IcH (unpublished
    In other studies, however, IP 3 injection or activation             data). In addition, the activation of Ca influx may re-
of PLC-coupled receptors induced two Ca-activated C1                    quire that the internal stores b e c o m e sufficiently de-
currents, but only the first c o m p o n e n t was indepen-             pleted. We have observed that with oocytes f r o m some
d e n t of Ca,, (Parker et al., 1985; Parker and Miledi,                                   2
                                                                        donors, Ic1_ induction is very small ( ~ 100 nA). If such
1987; Miledi et al., 1989; DeLisle et al., 1992; Lupu-                  an oocyte is placed in 0-Ca solution for :'-30 s and then
Meiri et al., 1993; Petersen and Berridge, 1994). T h e                 r e t u r n e d to normal Ca, Icl.2 becomes m u c h larger ( ~ 1
second c o m p o n e n t which was called Ti, and first de-             I~A) the instant the oocyte is r e t u r n e d to n o r m a l Ca.
scribed in detail by Parker et al. (1985) and Parker and                Thus, it seems that some oocytes are m o r e efficient at
Miledi (1987) requires Ca influx and is very likely the                 recycling the Ca that is released in response to IP.~ such
same as Ic>, described here. Parker et al. (1985) dem-                  that the pools do not b e c o m e depleted sufficiently to
onstrated that in some, but not all, native oocytes hy-                 activate Ca influx. Some oocytes may lose cytosolic Ca
perpolarization p r o d u c e d a small transient inward cur-           m o r e rapidly than others and this may d e t e r m i n e the
rent that increased with hyperpolarization and had a                                             z
                                                                        rate at which Icl_ develops. A n o t h e r possible consider-
duration of several seconds. These authors r e p o r t e d              ation is that oocytes may differ in the n u m b e r of follicu-
that this c u r r e n t was frequently labile and disappeared           lar cells a d h e r i n g to the oocyte. David Clapham (per-
after several minutes. A similar but m u c h larger current             sonal c o m m u n i c a t i o n ) has f o u n d that collagenase
was f o u n d in oocytes expressing receptors coupled to                t r e a t m e n t of oocytes does not always completely re-
phospholipase C when the receptors were activated by                    move the follicular cell layer. Thus, it is formally possi-

                                173      HARTZELL
ble that some of the currents we have described here                     was that the C1 current continued to increase when the
are due to adhering follicular cells that are attached by                m e m b r a n e was depolarized to arrest Ca influx (as in
gap junctions to the oocyte. At the level of a high-qual-                Fig. 7 B). Thus, they conclude that activating Ca must
ity dissecting microscope, we do not see any follicular                  come from stores. However, this interpretation de-
ceils, but a more detailed study would be required to                    pends on their assumption that the C1 current consists
adequately address this question.                                        of a single time- and voltage-independent c o m p o n e n t .
   Boton et al. (1989) have described two different Ca-                  However, we believe that the activating current u p o n
activated C1 conductances in oocytes permeabilized                       depolarization is the time- and voltage-dependent acti-
with A23187. When the permeabilized oocytes were                         vation of Ic~_l. This point of view is supported by the ob-
placed in solutions containing low Ca, only one kinetic                  servation that Icl. 2 is stimulated by Ca influx even when
c o m p o n e n t was observed, but addition of > 2 mM Ca                the Ca stores have been completely depleted by thapsi-
induced a current with two distinct kinetic compo-                       gargin and 0-Ca (Petersen and Berridge, 1994). It
nents. The IV curves for both c o m p o n e n t s determined             should be emphasized that this re-interpretation of the
by switching to Ca-containing solution at different                      regulation of C1 currents does not suggest that regener-
holding potentials or by voltage ramps were linear be-                   ative Ca release from IP~ sensitive pools does not exist
tween 0 and - 6 0 mV, but exhibited a decreasing con-                    u n d e r these conditions. However, the finding that Ca-
ductance at more negative potentials. This is similar to                 green fluorescence and C1 currents do not correlate
the steady-state 1V relationship of IcH described here,                  well (Yao and Parker, 1993) suggests that regardless of
but it remains uncertain how these two c o m p o n e n t s re-           the mechanisms generating the Ca transients, the C1
late to Icl.1 and Icl.Z. The two c o m p o n e n t s differed with       channels do not simply respond to bulk cytosolic Ca
respect to their sensitivity to Ca, block by injection of                concentration.




                                                                                                                                               Downloaded from www.jgp.org on February 20, 2005
EGTA, and extracellular application of the Cl-channel                       Petersen and Berridge (1994) suggest that the activa-
blocker 9-AC. Furthermore, the fast and slow compo-                      tion of the Icl.2-1ike current involves a positive feedback
nents were inactivated to differing extents by Ca via a                  mechanism, because the current induced by addition
mechanism that may involve phosphorylation by pro-                       of Ca to store-depleted oocytes produces a current that
tein kinase C (Boton et al., 1990; Petersen and Ber-                     increases explosively with voltage in the range between
ridge, 1994). The two c o m p o n e n t s of the current re-             - 5 0 and - 6 0 mV (see Fig. 4 C, in Petersen and Ber-
sponse to 5-HT or ACh could also be separated on the                     ridge, 1994). Their interpretation that this current has
basis of sensitivity to EGTA suggesting that these two                   a positive feedback depends on their assumption that
c o m p o n e n t s may be mediated by different channels or             the current observed in the voltage range of - 3 0 to 0
pathways.                                                                mV is the same current as that underlying the current
                                                                         in the range negative to - 3 0 mV. However, as we have
                                                                         shown, the current in the range positive to - 3 0 mV is
Mechanisms of I(:l_2Activation
                                                                         Ic~.1 whereas ICI_2D activates only negative to - 4 0 inV.
Yao and Parker (1993) have suggested that the Tin cur-                   O u r interpretation is that I~:l_2 is a voltage-dependent
rent, which we propose is identical to I{:1_2, arises be-                current that activates steeply with hyperpolarization in
cause Ca influx triggers regenerative Ca release from                    this range of potentials (Fig. 9 B).
IP~-sensitive stores. It is known that Ca and IP~ act syn-                  In summary, there are two Ca-activated C1 currents in
ergistically in stimulating Ca release from IP3-sensitive                Xenopus oocytes. One of these is stimulated preferen-
stores (Iino, 1990; Bezprozvanny et al., 1991). Yao and                  tially by Ca influx through store-operated channels,
Parker (1993) used several pieces of data to support                     whereas the other can be activated by Ca released from
this hypothesis, but one important piece of evidence                     stores or by Ca influx.


        Dedicated to my lather, who taught me how to use my hands. He died the day I submitted the final version of this manuscript,
        June 3, 1996.
            I would like to thank Susan Mierergerd and Amber Rinderkneckt for preparing and injecting oocytes and Drs. Y. Hirayama,
        A. Ivanov, David Clapham, and Henry Lester for comments on the manuscript.
            Supported by National Institutes of Health grants HL50474 and HI21195.
        Original version received 16 April 1996 and acceptedversion received5June 1996.

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