KCNQM-currents contribute to the resting membrane…

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					J Physiol 575.1 (2006) pp 175–189                                                                                                            175

KCNQ/M-currents contribute to the resting membrane
potential in rat visceral sensory neurons
Cynthia L. Wladyka and Diana L. Kunze
Rammelkamp Centre for Research and Education, MetroHealth Medical Centre and Department of Neurosciences, Case Western Reserve University,
Cleveland, OH 44109, USA

                The M-current is a slowly activating, non-inactivating potassium current that has been shown to
                be present in numerous cell types. In this study, KCNQ2, Q3 and Q5, the molecular correlates of
                M-current in neurons, were identified in the visceral sensory neurons of the nodose ganglia from
                rats through immunocytochemical studies. All neurons showed expression of each of the three
                proteins. In voltage clamp studies, the cognition-enhancing drug linopirdine (1–50 μM) and its
                analogue, XE991 (10 μM), quickly and irreversibly blocked a small, slowly activating current
                that had kinetic properties similar to KCNQ/M-currents. This current activated between −60
                and −55 mV, had a voltage-dependent activation time constant of 208 ± 12 ms at −20 mV, a
                deactivation time constant of 165 ± 24 ms at −50 mV and V 1/2 of −24 ± 2 mV, values which
                are consistent with previous reports for endogenous M-currents. In current clamp studies,
                these drugs also led to a depolarization of the resting membrane potential at values as negative
                as −60 mV. Flupirtine (10–20 μM), an M-current activator, caused a 3–14 mV leftward shift
                in the current–voltage relationship and also led to a hyperpolarization of resting membrane
                potential. These data indicate that the M-current is present in nodose neurons, is activated at
                resting membrane potential and that it is physiologically important in regulating excitability by
                maintaining cells at negative voltages.
                (Resubmitted 10 May 2006; accepted 14 June 2006; first published online 15 June 2006)
                Corresponding author D. L. Kunze: Rammelkamp Centre for Research and Education R326 MetroHealth Medical
                Centre, 2500 MetroHealth Drive, Cleveland, OH 44109-1998, USA. Email: dkunze@metrohealth.org

The visceral sensory neurons are critical in relaying                            of this stability is not completely known. At the resting
information from peripheral sensory receptors to the                             membrane potential, the hyperpolarization-activated,
nucleus of the solitary tract (NTS) in the brainstem. It                         cyclic-nucleotide-gated (HCN) family of ion channels
is there that visceral sensory information from cardio-                          is active, driving the membrane potential toward the
vascular, respiratory and gastrointestinal afferents is                          reversal of about −35 mV. This depolarizing drive is
integrated to generate the neural outflow that is essential                       offset by undetermined potassium current(s) (Doan &
for maintaining appropriate blood pressure, heart rate,                          Kunze, 1999). In this study we sought to identify the
respiration and gastrointestinal function. The afferent                          potassium current contributing to the resting membrane
pathways that relay this information have particular                             potential. A potential candidate which had not previously
characteristics that are unique for this purpose, including                      been identified in these cells was the M-current (I M ).
an axonal pathway that provides a direct transmission from                       The M-current is a slowly activating, non-inactivating,
the peripheral sensory receptor to the brainstem with a                          voltage-dependent potassium (K+ ) current that activates
single side branch to the cell soma.                                             in the range of the resting membrane potential. It acts
    Under physiological conditions, the soma of the                              to maintain the membrane potential below the threshold
sensory neurons are generally thought not to contribute                          for sodium current activation, thereby regulating
information to the activity arising at their peripheral                          neuronal firing and excitability (Marrion, 1997). Recent
receptor terminals, but simply to monitor the activity as it                     work has suggested that the M-current is carried by three
travels to the central terminations of the axons in the NTS.                     members of the KCNQ family of ion channel proteins,
It is essential then that the soma maintain a stable, negative                   KCNQ2, Q3 and Q5 (Wang et al. 1998; Lerche et al. 2000;
resting membrane potential (−50 to −60 mV) to ensure                             Schroeder et al. 2000; Wickenden et al. 2001; Shah et al.
that they fire only when sensory information from the peri-                       2002; Roche et al. 2002). These subunits have a relatively
pheral receptor terminal is received. The underlying basis                       restricted distribution in that they are primarily confined

C   2006 The Authors. Journal compilation   C   2006 The Physiological Society                                   DOI: 10.1113/jphysiol.2006.113308
176                                             C. L. Wladyka and D. L. Kunze                                                      J Physiol 575.1

to the nervous system. The importance of these channels         primary antibodies used were guinea pig anti-PGP at
in maintaining cells at resting potential is illustrated by     1 : 500 (Antibodies Inc.), rabbit anti-KCNQ2 at 1 : 200
the observation that mutations in KCNQ2 and KCNQ3               (Chemicon), rabbit anti-KCNQ3 at 1: 2000 (Chemicon)
play a role in causing benign familial neonatal convulsions     and rabbit anti-KCNQ5 at 1 : 500 (Chemicon). Antibody
(BFNC), a form of epilepsy (Jentsch, 2000).                     solutions were made in PBS with 1% BSA and incubated
   In the current study we have identified the presence          overnight at 4◦ C. One coverslip of cells was incubated
of KCNQ2, Q3 and Q5 in rat nodose ganglion cells.               with only PBS containing 1% BSA to serve as a negative
Using the known M-current blockers, linopirdine and             control. Cells were washed four times for 10 min each
XE991, we isolated an M-like current from other endo-           in PBS then incubated in secondary antibody solution
genous potassium currents. The current was slowly               containing PBS, 1% BSA, 10% NDS, donkey anti-rabbit
activating and non-inactivating with properties similar to      RedX at 1 : 300 (Jackson), and donkey anti-guinea pig
those of M-current and its suppression by the blockers          FITC at 1 : 500 (Jackson) for 1.5 h at room temperature
was irreversible over 10 min. In current clamp studies,         in the dark. Cells were washed four times for 10 min in
application of the blocker XE991 caused a depolarization        PBS then coverslipped with vectashield containing DAPI
of resting membrane potential. To further demonstrate the       (Vector). Images were obtained using a Nikon Eclipse E600
presence of M-current, we showed that application of the        microscope and a SPOT camera with SPOT advanced
M-current enhancer, flupirtine, increased current in these       software (Diagnostic Instruments, Inc.).
cells, led to a hyperpolarization of the resting membrane
potential, and caused a leftward shift in the current–voltage
relationship that is characteristic of M-current in the pre-    Immunohistochemistry
sence of this drug.
                                                                Adult ganglia were removed from 4-week-old rats, placed
                                                                in OCT tissue embedding media (Tissue-Tek) and quick
Methods                                                         frozen in 2-methylbutane. Frozen tissue was sectioned
                                                                at 6 μm and collected on Superfrost microscope slides
Isolation and culture of nodose ganglia                         (Fisher). Slides were post-fixed in 4% paraformaldehyde in
Neonatal (postnatal days 0–2) Sprague-Dawley rats               0.1 m phosphate buffer for 30 min. Slides that were going to
were asphyxiated by CO2 inhalation and the nodose               be stained with KCNQ3 and KCNQ5 were incubated with
ganglia were extracted in accordance with the                   warm citrate buffer for 10 min, while slides for KCNQ2
Case Western Reserve University Animal Research                 were rinsed in PBS. Staining procedures were the same as
Committee guidelines. The ganglia were collected in cold        those used for immunocytochemistry on the dissociated
Nodose Complete Media (NCM : DMEM-F12, (Gibco)                  cells with the following exceptions: all solutions contained
supplemented with 5% fetal bovine serum (HyClone) and           0.3% Triton X-100 (Sigma); PGP was not used; and the
1% penicillin–streptomycin–neomycin antibiotic mixture          secondary antibody for the KCNQ proteins was donkey
(Gibco)) then incubated for 30 min at 37◦ C in Earle’s          anti-rabbit FITC at 1 : 500 (Jackson).
Balanced Salt Solution (ICN) containing 1 mg ml−1
collagenase type 2 (Worthington). The enzyme solution
was then replaced by 37◦ C NCM containing 1.5 mg ml−1           Electrophysiology
albumin (bovine; Sigma) and the tissue was triturated           Electrophysiological experiments were performed on
with a fire-polished pipette to dissociate the cells. The        nodose neurons at room temperature or 35◦ C, 24–48 h
cell suspension was then plated into 35 mm Petri dishes         after plating. Using a whole-cell patch configuration
containing poly d-lysine-coated glass coverslips. The cells     under voltage- or current-clamp conditions, data were
were used within 48 h of plating.                               obtained with an Axopatch-1C patch clamp apparatus then
                                                                digitized and analysed using pCLAMP programs (Axon
                                                                Instruments) and Origin 7.5 (OriginLabs). Electrodes
                                                                (2–4 M ) were prepared from 8161 glass (Garner Glass).
Neonatal nodose ganglia were harvested and plated as            The extracellular solution for voltage-clamp experiments
described above. After 4 h in culture, the cells were rinsed    contained (mm): 140 N -methyl-d-glucamine, 5.4 KCl
twice for 5 min in PBS then fixed in 4% paraformaldehyde         (2.5 KCl was used for deactivation experiments only), 1
in 0.1 m phosphate buffer for 15 min. After another short       MgCl2 , 0.02 CaCl2 , and 10 Hepes, pH adjusted to 7.3
rinse in PBS, the cells were permeabilized in a 0.01%           with HCl. 4-Aminopyridine (4-AP; 5 mm) was added
saponin solution for 10 min. Following permeabilization,        to this bath solution in all voltage-clamp experiments
the cells were blocked for 30 min in PBS containing 1%          to block numerous K+ currents not pertinent to our
bovine serum albumin (BSA; w/v; Jackson) and 10%                studies. N -methyl-d-glucamine does not exhibit any
normal donkey serum (NDS; v/v; Jackson). Commercial             permeability through M channels (Block & Jones, 1996).

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J Physiol 575.1                                          KCNQ/M-currents in visceral sensory neurons                                     177

The extracellular solution for current-clamp studies                             Results
contained (mm): 137 NaCl, 5.4 KCl, 1 MgCl2 , 2 CaCl2 , 10
glucose, and 10 Hepes, pH adjusted to 7.3 by NaOH. The                           Immunocytochemical analysis of isolated neonatal nodose
pipette solution for voltage- and current-clamp contained                        ganglia cells in culture revealed the presence of KCNQ2,
(mm): 145 potassium aspartate, 2.2 EGTA, 10 Hepes, and                           Q3 and Q5 (Fig. 1, top row; left, centre and right columns,
1 MgCl2 , pH adjusted to 7.2 by KOH. Stock solutions                             respectively). The neuronal marker PGP was used to
of linopirdine (Sigma), XE991 (Tocris) and flupirtine                             identify neurons in the culture (Fig. 1, second row) and
(Sigma) were made in ethanol, distilled water and dimethyl                       distinguish them from other surrounding support cells.
sulfoxide, respectively, and were stored at −20◦ C until                         Co-localization of KCNQ and PGP labelling (Fig. 1, third
they were further diluted in bath solution and perfused                          row) showed all three of the KCNQ proteins tested were
onto cells in voltage- and current-clamp experiments.                            present in all neurons in the culture. We also used a second
The final concentration of the ethanol or DMSO was less                           set of antibodies targeted against KCNQ2, Q3 and Q5
than 0.2%. All values are mean ± s.e.m. unless otherwise                         (raised in goat, Santa Cruz Biotechnologies) on cultured
noted. Maximum series resistance errors were estimated                           cells that produced the same staining patterns as those in
to be < 4 mV for M-current recordings (range of series                           Fig. 1, supporting the observation that all three proteins
resistance was 4.4–11.7 M and was not compensated).                              are present in nodose cells under these conditions and at
HEK cells which stably expressed Kv2.1 (Kirsch et al.                            this developmental stage.
1991) were provided by A. Brown (MetroHealth Medical                                We also performed immunohistochemistry for KCNQ2,
Centre).                                                                         Q3 and Q5 on sections of whole nodose ganglia (Fig. 1,

                   Figure 1. The immunofluorescence localization of KCNQ2, KCNQ3 and KCNQ5
                   The top row shows the immunoreactivity of KCNQ2 (left), KCNQ3 (centre) and KCNQ5 (right) in isolated neonatal
                   nodose neurons, cultured on glass coverslips. The second row shows immunolabelling for the neuronal marker
                   PGP. The images in the top and second rows were digitally recorded as individual images and then merged to
                   give the images in the third row, with the yellow label indicating overlap between KCNQ and PGP. The fourth
                   row illustrates immunoreactivity of KCNQ2 (left), KCNQ3 (centre) and KCNQ5 (right) in sections of adult nodose
                   ganglia. Arrows indicate processes from pericytes surrounding the neurons. Scale bar is 20 μm for all images.

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178                                            C. L. Wladyka and D. L. Kunze                                                      J Physiol 575.1

bottom row) and on dissociated cells in culture from adult     However, we were able to eliminate this current in the
rats, to assess changes that may occur during development.     subpopulation where it was expressed because its block
As can be seen in Fig. 1, all three KCNQ proteins were still   was rapidly reversible (within 2 s of washing), while
present at 4 weeks of age. However, it can be seen more        block of the M-current was not reversible over the time
clearly at this age that there was variability of expression   course of the experiment (a similar irreversibility of
among cells, especially with KCNQ3 and Q5. KCNQ2               M-current over this time course was also noted in pre-
and Q5 were present throughout the entire cell, while          vious studies by Schnee & Brown, 1998; Wickenden et al.
KCNQ3 appeared to be more confined to the endoplasmic           2000, 2001; Yue & Yaari, 2004; Yeung & Greenwood, 2005).
reticulum and Golgi apparatus. Interestingly, KCNQ3 was        Thus, we obtained the irreversibly blocked component
also present in the fibres and the pericytes around the cells   representing the M-current by subtracting the current
(arrows in Fig. 1, bottom row, centre).                        recorded during the wash period from the control current
                                                               prior to administration of the drug. An example of this
Separation of M-current from other endogenous
                                                               current isolation method is shown in the top half of
                                                               Fig. 2. Currents were recorded from nodose neurons in
potassium currents
                                                               control solution (Fig. 2A) and in the presence of 50 μm
We used patch-clamp techniques to isolate the M-current        linopirdine (Fig. 2B). Upon washing out the drug with
which is generally a small component of total outward          control solution (Fig. 2C), only part of the current block
potassium current (Brown & Yu, 2000). Since M-current          was reversible. Figure 2D was obtained by subtracting B
is poorly blocked by 4-AP (Robbins, 2001), we added            from A and represents the total blocked current. By sub-
this drug at 5 mm to all of our voltage-clamp solutions        tracting C from A, we obtained the irreversibly blocked
to block the majority of the other potassium currents          current in Fig. 2E. Finally, the reversibly blocked current,
present in nodose neurons, and facilitate isolation of the     Fig. 2F, is the subtraction of B from C. The total current
KCNQ current. The currents present in nodose neurons           blocked in Fig. 2D had both a fast component as well
blocked by 4-AP included Kv1.1, Kv1.2, Kv1.6 (Glazebrook       as a slower M-like current that increased over the entire
et al. 2002) and Kv1.3, Kv1.5, Kv3.4 (unpublished data).       pulse. The slower current was more readily apparent as
We then used the M-current blockers, linopirdine and           the irreversibly blocked current (Fig. 2E), while reversibly
its analogue XE991, to isolate the KCNQ current from           blocked component is noticeably faster (Fig. 2F). The
the remaining 4-AP-insensitive current (Aiken et al. 1995;     reversibly blocked current had activation time similar to
Lamas et al. 1997; Costa & Brown, 1997; Schnee & Brown,        Kv2.1 (τ = 20–40 ms at 0 mV). Using HEK cells induced
1998). However, while these drugs are more selective for       to stably express Kv2.1 we confirmed the block of this
M-current, they have been shown to affect other currents       channel by XE991, and more so by linopirdine, in a readily
(Schnee & Brown, 1998; Wang et al. 1998) and we found          reversible manner (bottom half of Fig. 2). The series of
they affected two other currents in nodose neurons, in         four recordings in Fig. 2G represents currents in control
addition to M-current, that were still present due to their    solution, in the presence of 10 μm linopirdine, during a
low sensitivity to 4-AP.                                       washout with control solution, and finally the blocked
   The first additional current blocked was transient (a        Kv2.1 current obtained by subtracting the second panel
Kv4.3-like current) and present in a subpopulation of the      (+ Linopirdine) from the third panel (Wash). The series
neonatal neurons at P0 and in most of the neurons by P3.       in Fig. 2H is the same as in Fig. 2G except 10 μm XE991 was
Although the block by XE991 and linopirdine was weak, as       used instead of linopirdine. We fitted the time course of
previously reported (Wang et al. 1998), it interfered with     the subtracted currents with a standard mono-exponential
M-current at the beginning of depolarizing steps positive      function with a tau of activation in the presence of
to −20 mV, its activation threshold, making analysis of        linopirdine of 29 ± 4 ms (n = 8) and a tau in the
M-current activation over a range of voltages difficult.        presence of XE991 of 31 ± 5 ms (n = 8). Figure 2I
For that reason we evaluated the activation of the M-like      illustrates suppression of Kv2.1 by varying concentrations
current at −20 mV and/or, over a wider range, primarily        of XE991 and linopirdine, as compared with a
in P1 cells where the transient current was either absent or   representative control recording. Figure 2J is the
only minimally developed.                                      quantification of these data as normalized current.
   The second additional current blocked by linopirdine        In Fig. 2I and J it can be seen that 10 μm XE991
and XE991 was a sustained current, like M-current, but         suppressed the current by 6.5 ± 0.6% (n = 12), while
was one that activated much faster. This 4-AP-insensitive      10 μm linopirdine suppressed the current by 7.0 ± 1%
current was present in a subpopulation of neurons and, in      (n = 6). At 50 μm linopirdine, the current was blocked
those neurons, presented a problem in studying M-current       by 17.8 ± 2.5% (n = 6). At the high concentration of
because, at potentials positive to −30 mV, it was active       100 μm, XE991 blocked Kv2.1 by 20.4 ± 0.8% (n = 5) and
over the entire time course that M-current was activated.      linopirdine suppressed the current by 34.2 ± 4.5% (n = 4).

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J Physiol 575.1                                          KCNQ/M-currents in visceral sensory neurons                                      179

                   Figure 2. Effects of linopirdine and XE991 on 4-AP-insensitive currents in nodose neurons and on Kv2.1
                   currents in HEK cells
                   Top half: currents were evoked from nodose neurons in the presence of 4-AP by the protocol in the upper right
                   corner of the figure (800 ms pulses to voltages between −80 mV to +40 mV in 10 mV increments from a holding
                   potential of −80 mV, followed by a step to −30 mV for 150 ms). A illustrates the 4-AP-insenstive currents in
                   control solution. These currents decreased in the presence of 50 μM linopirdine (B) and partially recovered upon
                   washout of the drug (C). The total current blocked in D was obtained by subtracting B from A, while the irreversibly
                   blocked current (E) resulted from the subtraction of C from A. The component reversibly blocked by linopirdine (F)

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180                                                    C. L. Wladyka and D. L. Kunze                                                          J Physiol 575.1

               Figure 3. Effects of XE991 on activation of M-current in nodose neurons
               Experimental data illustrating current recorded in nodose neurons elicited by the protocol shown in the inset
               (an 800 ms pulse from a holding potential of −80 mV to a step voltage between −80 and +10 mV in 10 mV
               increments and finally back to −40 mV for 150 ms) in the absence (A) and presence (B) of 10 μM XE991 followed
               by a drug wash-out with bath solution (C). The irreversible XE991-sensitive current (D) was obtained by subtracting
               C from A to eliminate any reversible currents blocked by the drug which have been attributed to reversible block
               of Kv2.1. Activation time constants were determined from mono-exponential fits of the incremental pulses from
               −80 mV to the step voltages. The first 50 ms of the pulse were ignored during fitting due to the delay in M-current
               activation. The inset above D shows the mono-exponential fit (black line) overlaid on the original traces (grey) for
               steps to −20, 0 and 10 mV. E illustrates the activation curve of the data such as that in D, obtained from the tail
               current measurements (n = 15).

Pharmacological study of activation and deactivation                       concentration was low enough that it blocked less than 7%
of M-like current                                                          of the Kv2.1 present (see Fig. 2 for concentration–response
When isolating M-current, we preferentially used XE991                        Figure 3 shows a representative example of a cell without
over linopirdine because XE991 is more potent on                           Kv2.1-like current, as illustrated by the persistent XE991
M-current (Wang et al. 1998) and less potent on Kv2.1                      block even after returning to control solution (Fig. 3C).
(Fig. 2I and J). IC50 values for XE991 on M-currents                       Figure 3A shows a substantial slowly activating current
are reported in the range of 1 μm (Wang et al. 1998).                      that was recorded during perfusion with bath solution and
Since M-current in nodose is a small current we used                       evoked by a long depolarizing pulse in 10 mV incremental
10 μm XE991 (as did Wang et al. 1998; Shah et al. 2002;                    steps from −80 to +10 mV, followed by a step back to
Martire et al. 2004; Gu et al. 2005) to ensure that we                     −40 mV (see diagram in Fig. 3). XE991 (10 μm) reduced
blocked a majority of current to facilitate analysis. Also, this

               was isolated by subtracting B from C. Bottom half: the series of experimental data illustrate examples of current
               elicited by the protocol in the lower right corner of the figure (800 ms pulses to voltages between −80 mV to
               +10 mV in 10 mV increments from a holding potential of −80 mV, followed by a step to −40 mV for 150 ms). For
               10 μM linopirdine (G) and 10 μM XE991 (H), the first panel shows the currents during perfusion with bath solution
               (control), the second panel is in the presence of the drug, and the third panel is upon washing with bath solution
               (wash). The final panel in G and H was obtained by subtracting drug (second panel) from wash (third panel) and
               represents the Kv2.1 current blocked by the drug. I shows a compilation of Kv2.1 current suppression by 10, 50
               and 100 μM linopirdine and 10 and 100 μM XE991, as compared with a representative control recording, during
               a step from a holding potential of −80 mV to +20 mV for 800 ms then back to −40 mV for 50 ms. J shows the
               quantification of the data shown in I as normalized current for the control and each drug concentration.

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J Physiol 575.1                                          KCNQ/M-currents in visceral sensory neurons                                        181

the current by 35% rapidly within 1 min of application                           (Fig. 4B) by 37% from its level in control solution (Fig. 4A).
(Fig. 3B). The current then continued to decrease slowly                         The wash did not lead to current recovery (Fig. 4C), and
as more channels were bound by XE991. For this reason,                           in fact, decreased slightly, as was also seen in Fig. 3C.
upon washing (Fig. 3C), the current continued to decrease                        Again, using the control (Fig. 4A) minus wash (Fig. 4C)
briefly before control solution removed the remaining                             subtraction method to isolate the irreversibly blocked
XE991 from the bath. The slow time course of XE991                               M-current, we obtained the subtracted current in Fig. 4D.
block has been previously reported (Martire et al. 2004;                         For deactivation to −50 mV, we obtained a time constant
Gu et al. 2005; Peters et al. 2005; Yeung & Greenwood,                           of 165 ± 24 ms (n = 5), a value consistent with previously
2005). We were not, in all cases, able to maintain a                             reported M-current deactivation times (Brown & Adams,
recording for the time it took to achieve steady-state block                     1980; Wickenden et al. 2000). By using the depolarization
(approximately 10 min) and still complete the subsequent                         steps to −20 mV at the end of each pulse, we were able to
protocols. Therefore, we chose to examine the current after                      get another measurement of the activation time constant
the initial rapid blocking phase. The subtracted current in                      as being 180 ± 9 ms (n = 5), a value similar to our earlier
Fig. 3D was obtained by subtracting Fig. 3C from Fig. 3A.                        report of 208 ms.
This current has the characteristic shape of M-current with                         Two activators of M-current are useful for defining the
slow activation and lack of inactivation over the long pulse.                    current. Retigabine, and its functional, but less potent
Activation for this current consisted of two parts: an initial                   analogue flupirtine, exhibit potential as anti-convulsant
delayed rising phase (approximately 50 ms) followed by a                         drugs because of their ability to activate M-current,
slower activation phase (Main et al. 2000 and see Fig. 3                         and thus reduce excitability (Rundfeldt & Netzer, 2000;
legend). By ignoring the initial rising phase, we were able                      Wickenden et al. 2000, 2001; Tatulian et al. 2001; Passmore
to fit the slow activation with a single exponential function,                    et al. 2003; Martire et al. 2004). In voltage clamp, applying
as has been done for other potassium currents exhibiting                         flupirtine led to an increase in overall current when applied
an early delayed rising phase, such as Kv2.1 (Klemic et al.                      to nodose neurons, as can be seen in Fig. 5. Application of
1998). The current had a voltage-dependent activation                            20 μm flupirtine (Fig. 5B) caused a 20% increase in current
time, with a value of 208 ± 12 ms at −20 mV (n = 11) and                         from control levels (Fig. 5A) and was readily reversible
102 ± 11 ms at +10 mV (n = 13). We were able to fit these                         upon washing (Fig. 5C). By subtracting the control current
currents without interference from the 4-AP-insensitive                          (Fig. 5A) from the current in the presence of flupirtine
transient current discussed earlier because we limited                           (Fig. 5B), we obtained the current that increased due to this
our studies to primarily age P1 cells, and in a few P2                           drug (Fig. 5D). This current had the characteristic shape
neurons, where the transient was minimally developed.                            and slow activation of M-current and showed saturation
The activation curve (Fig. 3E) was compiled from data                            above −10 mV. The current–voltage relationship seen in
from 15 cells and was plotted using the amplitude of                             Fig. 5E was compiled from the normalized data of seven
tail currents of the subtracted current (like those in                           cells. It illustrates a 7–14 mV leftward shift in activation in
Fig. 3D) produced immediately upon stepping back to                              the presence of flupirtine across the entire range in which
−40 mV after the end of the pulse. A small current was                           M-current is activated, −60 to +40 mV, much like that
present at the typical resting membrane potential, −65 mV                        seen in previous studies with flupirtine (Martire et al.
to −60 mV, which is characteristic of M channels. All                            2004; Wu & Dworetzky, 2005) but less than that seen
channels appeared to be open by 0 mV, with the majority                          with the more potent analogue retigabine (Wickenden
opening between −40 mV and −10 mV. Using a single                                et al. 2000, 2001; Tatulian et al. 2001; Martire et al.
Boltzmann model, the activation curve was fitted with a                           2004). Results obtained for 7 out of 10 cells in the
half-activation voltage of −24 ± 2 mV, a value consistent                        presence of 20 μm flupirtine had an average leftward shift
with previous reports (Tinel et al. 1998; Tatulian et al.                        of 10.1 ± 0.9 mV, and 4 out of 7 cells in the presence of
2001; though the range of reported values varies between                         10 μm flupirtine showed a leftward shift with an average
−8 to −44 mV) and a slope factor of 8.4 ± 1.6. Similar                           of 5.3 ± 0.9 mV. In the remaining six neurons we were
slope values were reported by Wang et al. (1998), Tinel                          unable to resolve changes in the M-current because of
et al. (1998) and Roche et al. (2002) (and others reported                       small simultaneous changes in the large Kv2.1 component.
values varying widely between 5.5 and 18.6).                                     Figure 5F illustrates concentration–response data points
   M-currents are typically compared based on the time                           for flupirtine on neonatal nodose neurons. The average
course of the deactivation current. To investigate this                          current increases during a step to −50 mV from baseline
property, we used a standard M-current protocol (similar                         levels at −60 mV were plotted for concentrations between
to Adams et al. 1982) in which the current was activated                         1 and 100 μm. By using a voltage step at a negative potential
by holding cells at −20 mV for 1 s, then deactivated                             where other voltage-gated potassium channels are inactive,
by sequential, 10 mV hyperpolarizing steps, and finally                           we were able to ensure that our measurements were limited
depolarized again to −20 mV (see diagram in Fig. 4). The                         to M-current. However, since the amplitude of M-current
presence of 10 μm XE991 reduced the deactivation current                         was so small at this voltage, we were unable to resolve small

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182                                                  C. L. Wladyka and D. L. Kunze                                                           J Physiol 575.1

changes that may have occurred at concentrations lower                   below the threshold for activation of M channels (n = 5).
than 1 μm. For this reason, it was not possible to determine             Figure 6C and D illustrate representative examples of the
a single IC50 value.                                                     effects of 20 μm flupirtine on a cell with a resting potential
                                                                         of −58 mV (Fig. 6C) and a cell with a potential of −65 mV
Pharmacological effects on resting
                                                                         (Fig. 6D). The effect of flupirtine (20–40 s) in Fig. 6C was a
                                                                         rapid, 6 mV hyperpolarization, that readily reversed upon
membrane potential
                                                                         washing (n = 14, 7.0 ± 0.6 mV, range 5–11 mV). No effect
M-currents are unique among voltage-gated K+ currents                    was seen from flupirtine in Fig. 6D, when the potential
in neonatal nodose neurons in that they activate at,                     was below the threshold of M-current activation (n = 3).
or slightly more negative than, the resting membrane                     Also in current-clamp configuration, we evoked action
potential (approximately −60 mV). This property was                      potentials in cells by injecting depolarizing current pulses
evident in the activation curve in Fig. 3E, but to                       (typically 5–20 pA). As seen in Fig. 6F, application of
further investigate the activity of these channels near the              20 μm flupirtine (20–50 s) caused a hyperpolarization of
resting membrane potential, we employed current-clamp                    the membrane potential from −41 mV to −49 mV. This
experiments. Membrane potentials were continuously                       hyperpolarization was large enough that applying the same
recorded from cells at rest. Figure 6A and B show the effects            depolarizing current step (15 pA) was no longer able to
of 10 μm XE991 on two representative cells: one with a                   evoke action potentials. Similar results were obtained in
resting potential of −56 mV (Fig. 6A) and the other with                 three other cells. This effect was fully reversible after only
a more negative resting potential of −71 mV (Fig. 6B).                   10 s of washing.
Figure 6A demonstrates a slow depolarization when XE991                     In most of the cells, adding XE991 to inhibit M-current
was applied (20–50 s) and a lack of immediate reversibility              caused a depolarization that was not large enough on
upon washing (50–80 s). In 14 cells the mean change was                  its own to trigger action potential generation. However,
3.6 ± 0.4 mV (range 1–6 mV). Figure 6B illustrates the                   in a subset of neurons when a depolarizing current step
absence of any response to XE991 at this very negative                   was used to elicit an action potential, application of
membrane potential, indicating that this cell was resting                10 μm XE991 increased the number of spikes produced in

              Figure 4. Effects of XE991 on deactivation of M-current in nodose neurons
              Experimental data illustrating an example of deactivation currents evoked by the protocol illustrated in the inset
              (hyperpolarizing the cells from a holding potential of −20 mV to step voltages from −30 to −80 mV in 10 mV
              increments for 1000 ms each to deactivate the channels and then depolarizing back to −20 mV for 1000 ms to
              reactivate the M channels). This protocol was applied in the absence (A) and presence (B) of 10 μM XE991 and
              followed by a wash-out with bath solution after drug application (C). The irreversible XE991-sensitive current (D)
              was obtained by subtracting C from A. The inset above D shows the exponential fits (black lines) for deactivation
              to −40, −50 and −60 mV and activation to −20 mV overlaid on the original traces (grey).

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J Physiol 575.1                                          KCNQ/M-currents in visceral sensory neurons                                      183

response to the current pulse (Fig. 6G). Prior to application                       We also investigated the presence and function of
of XE991 (black trace), an 8 pA depolarizing pulse evoked                        M-currents in neurons at physiological temperature
only a single action potential from a cell that was resting                      (approximately 35◦ C) in neonatal cells, as opposed to room
at −56 mV. However, after application of XE991, the same                         temperature (22◦ C) where our other experiments were
pulse elicited four action potentials and the resting level of                   performed. In this case, XE991 produced a depolarization
the cell also depolarized to −45 mV. Similar results were                        of the resting membrane potential that was similar to that
seen in five additional cells where there was an increase                         seen in neonatal neurons at room temperature (n = 6,
from one spike to two to seven spikes and an average change                      4.33 ± 0.97 mV, range 2–8 mV). Also, flupirtine was noted
in resting potential of 7.2 ± 1.5 mV (range 3–12 mV).                            to have the same hyperpolarizing effect at physiological
   We tested for differential effects of XE991 and flupirtine,                    temperature (n = 9, 7.2 ± 1.1 mV, range 3–13 mV).
on A- and C-type nodose neurons. Using the presence of                              Next we tested the effects of XE991 and flupirtine on
tetrodotoxin-resistant sodium current to identify C-type                         the resting membrane potential of older neurons (from
neurons (as outlined by Schild & Kunze, 1997), we                                4-week-old animals) to assess functional developmental
distinguished between the two neuronal subpopulations                            changes that may have occurred. Again, we observed a
and observed the effect of XE991 and flupirtine on each                           similar depolarization due to XE991 (n = 5, 5.7 ± 1.1 mV,
group. In the presence of XE991, A-type neurons showed                           range 4–9 mV) and hyperpolarization due to flupirtine
a depolarization of 4.0 ± 1.0 mV (n = 6, range 3–8 mV)                           (n = 7, 6.7 ± 0.9 mV, range 4–11 mV) like that seen in
while C-type neurons depolarized 3.9 ± 0.4 mV (n = 6,                            neonatal neurons, indicating the continued presence and
range 3–5 mV). Upon exposure to flupirtine, A-type                                function of M-currents in adult animals.
neurons hyperpolarized by 3.3 ± 0.3 mV (n = 5, range                                Finally, we investigated the antagonistic effects of these
2–4 mV), in contrast to C-type neurons that showed                               two drugs to confirm that they were both producing their
6.7 ± 1.3 mV hyperpolarization (n = 5, range 4–11 mV).                           effects through the same channel family. Since slow block

                   Figure 5. Effects of flupirtine on M-currents in nodose neurons
                   Experimental data showing current elicited by the protocol (shown above C; an 800 ms pulse from a holding
                   potential of −80 mV to voltages between −100 and +40 mV in 10 mV increments, followed by a step to
                   −30 mV for 150 ms) that was applied in the absence (A), presence (B) and wash-out (C) of 20 μM flupirtine.
                   The flupirtine-sensitive current (D) was obtained by subtracting A from B. The current–voltage relationship (E) was
                   plotted as the voltage step versus the normalized current at 800 ms (near the end of the long pulse) from 7 cells
                   for the control data ( ) and data in the presence of flupirtine ( ). The flupirtine concentration–response data (F)
                   was compiled from the average current increases during a step to −50 mV from baseline levels at −60 mV for
                   cells in the presence of 1 μM (n = 8), 3 μM (n = 8), 10 μM (n = 7), 20 μM (n = 10), 30 μM (n = 14), and 100 μM
                   (n = 7) flupirtine.

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184                                                   C. L. Wladyka and D. L. Kunze                                                           J Physiol 575.1

is characteristic of XE991, we tested these antagonistic                  Effects of other channel blockers
properties by pre-treating cells with 10 μm XE991 for
                                                                          Although barium non-specifically blocks several K+
different lengths of time, then measuring the hyper-
                                                                          channels, it has been shown to block M channels as
polarizing effect of 20 μm flupirtine (Fig. 6E). Increasing
                                                                          well (Cuevas et al. 1997; Selyanko et al. 1999; Passmore
the time in XE991 prevented the action of flupirtine, as
                                                                          et al. 2003). The application of 5 mm barium reversibly
can be seen by the decrease in the hyperpolarization effect.
                                                                          suppressed a current with similar properties to those of
These data indicate that XE991 and flupirtine do exert their
                                                                          the current blocked by XE991 (data not shown).
effects on the same channels.

              Figure 6. Effect of XE991 and flupirtine on resting membrane potential
              Current-clamp recordings of the effect of 10 μM XE991 on representative cells with resting membrane potentials
              of −56 mV (A) and −71 mV (B). In A, XE991 was applied between 20 and 50 s while in B, XE991 was applied
              between 20 and 60 s. Current-clamp recordings of the effect of flupirtine on resting membrane potential are
              shown for cells at potentials of −58 mV (C) and −65 mV (D). Flupirtine (20 μM) was applied to the cells in both
              C and D between 20 and 40 s. Fresh bath solution was applied to cells at all times other than the intervals
              when the drugs were used. E illustrates antagonism between XE991 and flupirtine. The average hyperpolarization
              resulting from application of 20 μM flupirtine is plotted against the length of time the cells were pretreated with
              10 μM XE991 (n = 14, 4, 3, 4, 3 and 3 mV for pretreatment times of 0, 5, 8, 10, 12 and 15 min, respectively). In
              F, action potentials were evoked by injecting 15 pA of current for 9 s out of every 10 s segment. Flupirtine (20 μM)
              was applied between 20 and 50 ms. G illustrates an increase in action potential discharge in response to an 8 pA
              depolarizing pulse in the presence of 10 μM XE991 (grey trace), compared with control levels (black trace).

                                                                               C   2006 The Authors. Journal compilation   C   2006 The Physiological Society
J Physiol 575.1                                          KCNQ/M-currents in visceral sensory neurons                                               185

   We also tested the potassium channel blocker                                  in shaping the cell activity. The unique features of
tetraethylammonium (TEA) because it has been shown                               M-current and the availability of blocking and activating
previously to exert blocking effects on M-currents,                              agents have allowed us to identify an M-like current in
though to varying degrees depending on KCNQ                                      the nodose neurons. The common method of comparison
subunit composition (Hadley et al. 2000). To avoid                               of M-currents is typically the deactivation time constant
activating the other TEA-sensitive current, Kv2.1, we used                       obtained using an ‘M-current protocol’ (Adams et al.
a small voltage step at a negative potential (−60 mV to                          1982). Our deactivation time constant of 165 ± 24 ms at
−40 mV) to evoke a small M-current. We then applied TEA                          −50 mV is similar to numerous reported values including
at concentrations of 1 mm and 10 mm (a representative                            those of 150–200 ms (Brown & Adams, 1980), and 146 ms
example is shown in Fig. 7). Application of 1 mm TEA                             (Wickenden et al. 2000) (also Schnee & Brown, 1998;
(light grey trace) produced a very small change in holding                       Tinel et al. 2000; Gamper et al. 2003). Other groups have
current and the current activated by the voltage step                            reported the need to use a two exponential fit on their
from control levels (black trace), while 10 mm TEA (dark                         M-currents where the ‘fast’ value is similar to our 165 ms
grey trace) completely abolished the slowly activating                           value, and the ‘slow’ value is of the order of 800–900 ms
current. As the current in this range is generally only a                        (Wang et al. 1998; Lerche et al. 2000; Pan et al. 2001).
few picoamperes, it was not possible to construct a reliable                        Activation time constants show the same trend where
concentration–response curve at the lower concentrations.                        some activation curves are fitted with a single exponential
The main observation is that while 10 mm TEA produced                            (Brown & Adams, 1980; Adams et al. 1982; Tinel et al. 2000;
a strong block, 1 mm TEA did not. The latter would be                            Gamper et al. 2003) with values similar to those in the
expected to block KCNQ2 homomultimers. Similar results                           present study and others are fitted using two exponentials
were seen in seven neurons.                                                      in which one of the values is in the 100–200 ms range and
   The only other slow activating current that might                             the other is either faster or slower (Lerche et al. 2000; Pan
account for the current we observed is carried by erg                            et al. 2001). A number of explanations can be proposed to
channels which have also been shown to associate with                            account for differences in reported time constants. Studies
KCNQ currents (Selyanko et al. 1999; Meves et al. 1999).                         in expression systems such as Xenopus oocytes (Wang
However, astemizole, an erg/eag blocker (Ulens & Tytgat,                         et al. 1998; Lerche et al. 2000; Main et al. 2000; Schroeder
2000; Garcia-Ferreiro et al. 2004), had no effect on                             et al. 2000) and Chinese hamster ovary cells (Rundfeldt
the linopirdine- or XE991-sensitive current in nodose                            & Netzer, 2000; Selyanko et al. 2000; Wickenden et al.
neurons. Linopirdine has only a very weak blocking effect                        2000, 2001; Pan et al. 2001; Tatulian et al. 2001; Hadley
on erg/eag channels (IC50 > 30–85 μm, Wang et al. 1998;                          et al. 2003; Martire et al. 2004) provide useful information
IC50 > 20 μm, Meves et al. 1999) and XE991 is even less
potent (> 50–100 μm, Wang et al. 1998). Therefore, it
is unlikely that the current observed in these studies is
composed of erg/eag channels.

In this work we presented the first report of
M-currents, and their molecular correlates, the
KCNQ2/Q3/Q5 proteins, in visceral sensory neurons.
The cognition-enhancing drugs linopirdine and XE991
blocked a current with activation and deactivation
characteristics of M-current. XE991 depolarized the
membrane potential when its resting level fell within the
activation range of M-current. Finally, we showed that
flupirtine, the functional analogue of the anti-epileptic
drug retigabine, increased a current with M-like properties
in these cells and caused a hyperpolarization in resting
                                                                                 Figure 7. Effects of TEA on M-currents
membrane potential.
                                                                                 Representative example of currents evoked from nodose neurons by a
                                                                                 single step from −60 mV to −40 mV. Currents were recorded in
                                                                                 control solution (black trace) and in the presence of 1 mM TEA (light
Supporting evidence for M-current in nodose neurons                              grey trace) and 10 mM TEA (dark grey trace). Each trace is the average
                                                                                 of 10 recordings made under the specified condition (control, 1 mM
Separation of potassium currents in native cells is difficult                     TEA or 10 mM TEA) and noise was smoothed out by averaging
but essential to understanding the role of specific channels                      adjacent points.

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186                                               C. L. Wladyka and D. L. Kunze                                                      J Physiol 575.1

about the basic properties of the channels, but may not be        nodose cells consisted of distinct components. Reports
entirely representative of the characteristics of endogenous      of linopirdine affecting other currents, including delayed
channels where the specific combinations of subunits that          rectifier currents, have been published (Schnee & Brown,
contribute to the current in a particular cell are unknown,       1998), but no previous reports have specifically identified
as is the role of accessory subunits that may modulate the        Kv2.1. Kv4.3 has also been shown to be blocked by
kinetics (such as the KCNE family studied by Tinel et al.         linopirdine and XE991 (Wang et al. 1998). These studies
2000). In addition, it has been shown that KCNQ2 can              provide cautionary notes for attributing the effects of these
form functional splice variants with different activation         drugs to effects on endogenous M-currents in native cells,
and deactivation characteristics (Tinel et al. 1998; Pan et al.   particularly in current clamp experiments where the action
2001). Finally, our electrophysiological activation studies       potential duration, after-hyperpolarization and repetitive
focused mainly on neonatal sensory neurons. There are             discharge are compared with and without block.
reports of developmental changes in expression of specific
subunits which can therefore give rise to slight changes in
kinetics of M-current (Tinel et al. 1998; Shah et al. 2002;       Subunit composition
Hadley et al. 2003).                                              KCNQ2, KCNQ3 and KCNQ5 immunoreactivity was
   Our data provide two other pieces of evidence                  present at varying levels among the entire population of
supporting the presence of M-current in nodose neurons.           nodose neurons, as was the current with the characteristics
The first is the activity of this channel in the range of the      of the M-current which was present in both A- and
resting membrane potential (−50 to −60 mV). M-current             C-type neurons. It is not uncommon to see all three
is one of the few K+ channels that are active at voltages         neuronal KCNQ subunits present in a neuron (Shah
as low as −60 mV (Adams et al. 1982). Our activation              et al. 2002; Passmore et al. 2003). The three proteins
curve data showed that M-current in nodose activates              associate to form different homomultimers (Q2 or Q5)
between −70 to −60 mV, with V 1/2 of −24 mV, and our              or heteromultimers (Q2 + Q3, Q2 + Q5 and Q3 + Q5).
current-clamp studies also showed a depolarization from           Various KCNQ subunits can be distinguished from one
the resting membrane potential in the presence of M               another by different sensitivities to tetraethylammonium
channel blockers. We not only demonstrated this effect            (TEA) (Hadley et al. 2000; Lerche et al. 2000; Shah et al.
on the resting membrane potential of neonatal neurons,            2002; Passmore et al. 2003). As the M-current in the
both A- and C-type, but on neonatal neurons at physio-            neonatal nodose neurons was minimally affected at 1 mm
logical temperature, as well as on adult neurons. This            TEA, it is unlikely that the M-like current we observed is
characteristic of the current has important implications for      composed of many KCNQ2 monomeric channels, which
the role of M channels in maintaining membrane potential          are reported to have an IC50 for TEA block of 0.13–0.3 mm
below the threshold for action potential generation.              (Wang et al. 1998; Hadley et al. 2000; Shapiro et al. 2000;
   The second piece of additional evidence supporting the         Wickenden et al. 2000; Robbins, 2001). In fact, our TEA
presence of M-current in nodose cells is the increased            data suggest that the subunit composition is primarily
activation by flupirtine and the hyperpolarizing effect            KCNQ2/3 since TEA at 10 mm blocked the M-current
on membrane potential caused by this drug. Our data               observed during a step from −60 mV to −50 mV (7/7
in the presence of flupirtine display the characteristic           neurons). Block of homomultimers of KCNQ3 or KCNQ5
leftward shift in the current–voltage relationship as others      or heteromultimers of KCNQ3/5 would be expected at
have seen with flupirtine and the analogue retigabine              concentrations much higher than 10 mm (Wang et al.
(Wickenden et al. 2000; Tatulian et al. 2001; Martire et al.      1998; Hadley et al. 2000, 2003; Lerche et al. 2000;
2004) as well as the ‘saturation’ at higher voltage steps         Schroeder et al. 2000; Wickenden et al. 2000; Robbins,
(Tatulian et al. 2001). Furthermore, in current clamp 40/40       2001). In contrast to our results, the IC50 values for
neurons, including neonatal, adult and those recorded at          the response to TEA among nociceptive dorsal root
37◦ C, responded to flupirtine with a hyperpolarization of         sensory neurons fell into two groups, 0.2–0.6 mm and
the membrane potential, adding support to the immuno-             3.9–4.7 mm (Passmore et al. 2003), suggesting mixed
logical data which indicates the presence of M-current in         expression levels of the various subunits among cells.
all nodose neurons.                                               In superior cervical ganglion neurons, developmental
                                                                  increases in the expression of KCNQ3 and subsequent
Note of caution: use of linopirdine and XE991                     KCNQ2/3 heteromultimer formation are suggested to
                                                                  account for the shift from a two to one component TEA
to evaluate endogenous M-currents
                                                                  inhibition curve between P17 to P45 rats (Hadley et al.
Using HEK cells expressing Kv2.1, we demonstrated                 2003). Similarly, Shah et al. (2002) report two values for
the novel finding that linopirdine and XE991 block                 hippocampal cells, an IC50 of 0.7 mm, and a second one
Kv2.1 in a concentration-dependent manner, and this               of 1.4 mm (the latter produced only a partial block). At
observation explained why our blocked current in                  this time, we cannot rule out that we have missed a small

                                                                      C   2006 The Authors. Journal compilation   C   2006 The Physiological Society
J Physiol 575.1                                          KCNQ/M-currents in visceral sensory neurons                                       187

subpopulation of nodose neurons with a high sensitivity                          information that is transmitted to the central nervous
to TEA, reflective of KCNQ2 homomultimers. To what                                system from the peripheral receptor is to remain faithful
extent is there heterogeneity among neurons within the                           to the stimulus. Presently we have no information on the
ganglion? In this study we did not discern clear differences                     presence of M-current at the peripheral and central
among neurons with respect to the amplitude or kinetics                          terminals of the sensory afferents where modulation by
of the M-currents such as we and others have seen for HCN                        paracrine and neurocrine factors such as substance P,
(I h ) and tetrodotoxin-resistant sodium currents in nodose                      bradykinin and angiotensin II (for review see Brown &
neurons. While there are differences in the distribution of                      Yu, 2000) may play a role in sensory transduction or
KCNQ subunit immunoreactivity among neurons this is                              in synaptic transmission at the central terminals in the
not necessarily reflected in somal currents as previously                         nucleus of the solitary tract. Nor do we know which of
demonstrated by others. For instance, Passmore et al.                            the KCNQ subunits we have observed in the soma may be
(2003) demonstrated that KCNQ5 was present in both                               expressed in those regions. There is precedence for selective
large and small sensory neurons although the high TEA                            distribution of KCNQ subunits to non-somal regions.
sensitivity of the M-current did not support a role for this                     Devaux et al. (2004) have demonstrated expression of
subunit in the somal current. A similar situation exists                         KCNQ2 but not KCNQ3 at the nodal regions of myelinated
in hippocampal neurons (Shah et al. 2002). The role of                           nerves co-localizing with sodium channels and Martire
KCNQ5 in those neurons and in the nodose neurons of                              et al. (2004) have implicated KCNQ2 but not KCNQ3 in the
the present studies remains unknown. The only data in                            secretion of neurotransmitters from hippocampal nerve
the present study that suggest there may be differences in                       terminals.
the functional expression among the nodose neurons is the
wide spread in the current response values to flupirtine
at each concentration point of the dose–response curve,                          Potential role of visceral sensory afferents in benign
especially at the higher concentrations (> 20 μm). This                          familial neonatal convulsions
observation may indicate that the concentration–response                         KCNQ2, KCNQ3 and KCNQ5 subunits have a relatively
is not merely a single component and that these differences                      restricted distribution; they are primarily confined to the
may reflect the differing KCNQ subunit compositions of                            nervous system. They have been shown to be present in
the cells and potentially distinct flupirtine sensitivities of                    the hippocampus (Shah et al. 2002), in various cortical
specific subunit combinations. In fact, 2 of 7 neurons tested                     regions, cerebellum (KCNQ2 only), spinal cord (KCNQ2
at both 1 and 10 μm showed little or no further increase                         and KCNQ3) and in dorsal root ganglia (Passmore et al.
in current at 10 μm as compared with 1 μm, while the                             2003). Mutations in KCNQ2 and KCNQ3 underlie an auto-
other cells tested showed greater increases at 10 μm. This                       somal dominant form of epilepsy that appears in newborn
suggests that there may be a subpopulation of neurons                            infants, benign familial neonatal convulsions (BFNC). In
expressing channels with subunit combinations that are                           addition to convulsions, symptoms are reported to include
more sensitive to flupirtine. On the other hand, little                           apnoea and bradycardia in relation to the seizures (Hirsch
information is available on the concentration–response                           et al. 1993; Claes et al. 2004). Although not well studied, in
relationship of flupirtine on expressed KCNQ subunits. A                          at least one case apnoea is reported to precede the seizure
controlled expression study of the three neuronal KCNQ                           (Lerche et al. 1999). The presence of the KCNQ subunits
subunits may be a way to further investigate the effects                         in sympathetic neurons (Brown & Adams, 1980; Wang
of flupirtine, and separate concentration–response curves                         et al. 1998), intracardiac neurons (Cuevas et al. 1997) and,
might be necessary for each combination.                                         now, in visceral sensory neurons focuses attention on a
                                                                                 possible role of these subunits in symptoms related to the
                                                                                 cardio-respiratory reflexes.
Role of M-current in nodose neurons
In our studies we evaluated the role of the M-current in
regulating membrane potential. Although this current is                          Adams PR, Brown DA & Constanti A (1982). M-currents and
relatively small in relation to other currents present in                          other potassium currents in bullfrog sympathetic neurons.
nodose cells, it is active at the resting membrane potential                       J Physiol 330, 537–572.
                                                                                 Aiken SP, Lampe BJ, Murphy PA & Brown BS (1995).
where, within a very narrow range, the input resistance
                                                                                   Reduction of spike frequency adaptation and blockade of
in the neonatal neurons is high (≥ 1G ; Doan & Kunze,                              M-current in rat CA1 pyramidal neurons by linopirdine
1999). Therefore, changes in M-current of only a few                               (DuP 996), a neurotransmitter release enhancer.
picoamperes can have a significant effect on the resting                            Br J Pharmacol 115, 1163–1168.
membrane potential. The effects of XE991 and flupirtine                           Block BM & Jones SW (1996). Ion permeation and block of
on the membrane potential support this role. At the                                M-type and delayed rectifier potassium channels. J General
level of the soma, membrane stability is essential if the                          Physiol 107, 473–488.

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188                                                C. L. Wladyka and D. L. Kunze                                                       J Physiol 575.1

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  480, 137–141.                                                                  This work was supported by NIH HL25830 and HL61436.

C   2006 The Authors. Journal compilation   C   2006 The Physiological Society

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