Docstoc

University of Debrecen

Document Sample
University of Debrecen Powered By Docstoc
					             University of Debrecen
              Medical and Health Science Center
           Department of Biophysics and Cell Biology
           Research Centre for Molecular Medicine




Regulation of Kv1.3 activity in human T lymphocytes:
     peptide blockers and molecular interactions


                      György Panyi



                                      August 27, 2005, Montpellier
          Simple view of an ion channel:
a hydrophilic pore allowing very rapid ion movement
           across the membrane (108 /s)
                    heartbeat
    movement
                                   secretion

immunity

               Ion Channels
                                      memory

contraction
                               perception
           sensation thought
How do ion channels regulate the immune functions?

                                                 K+                       K+



                                      TCR/




                                                                         IKCa1
                                                 Kv1.3
                                      CD3

                                                          Em~-50 mV
                       IP3      PLC                                       CaM
           ER
                IP R
                  3


                                          Ca2+                CaM         Calcineurin

  CRAC                                                                           NF-AT
                                [Ca2+]
                                                                     IL-2 gene expression
                                         time                            proliferation

                                             T cell


Panyi et al., Trends Immunol., 2004, 25: 565-569;        Panyi et al., Imm. Lett., 2004, 92:55-66.
Ion channels are oligomeric transmembrane proteins

  Extracellular             α

                      α         α
                          α

  Intracellular

              voltage-sensing
                  domain      pore




              S1 S2
                      S3b       +        S6
                                    S5
                                +

                          NH2 COOH
          How do we study the channels?
By measuring ionic currents using the patch-clamp technique
                                                                      Rf

                                                           Ip                          Vout
                                                                            -
                                                                            +


                                                                            Vhold Op. amp
                                       Em            Im




                                                       +30 mV
                             600       +30 mV
                                                       -10 mV
                                                       -50 mV
              current (pA)




                             400
                                                  -80 mV

                             200
                                       -40 mV
                               0

                                   0        200      400        600   800       1000
                                                       time (ms)
                           How do K+ channels of T cells compare?
                                                                                 100
                                                                                 100
                                   +30 mV                                                        high [Ca2+]i




                                                                 current (pA)
               600
current (pA)


                                   -10 mV                                         50
                                                                                  50




                                                                current (pA)
               400                  -50 mV                                         0
                               -80 mV                                                                    low [Ca2+]i
                                                                                 -50
                                                                                 -50
               200
                                                                                -100
                                                                                -100

                 0                                                              -150
                                                                                -150

                     0   200      400        600   800   1000                       -140 -120 -100 -80
                                                                                   -140 -120 -100 -80    -60 -40
                                                                                                         -60 -40

                                  time (ms)                                            membrane potential (mV)
                                                                                       membrane potential (mV)

                                   Kv1.3                                                      IKCa1
Gating:                        voltage-gated                                            Ca2+-activated

Single channel
                                similar (~10 pS)                                        similar (~10 pS)
conductance

Selectivity                     K+ selective                                             K+ selective

Block                           different sensitivity to organic and inorganic compounds
             three important problems




How does each subunit contribute to gating, specifically slow inactivation?



What kind of molecules inhibit Kv1.3 and IKCa1 channels?



Can the microenvironment of the membrane alter the channels’ behavior?
                     How does each subunit contribute to gating,
                           specifically slow inactivation?

                                 WT                                                      Mut   Mut
               1.2                                                         0.4
current (nA)




                                                            current (nA)
               0.9                                                         0.3
                                                                           1.0
                             τi~ 200 ms                                                        τi~ 4 ms




                                                          current (nA)
               0.6                                                         0.2

               0.3                                                         0.1

               0.0                                                           0
                                                                           0.0

                     0            500              1000                          0              25           50
                                                                                     0     25    50
                             time (ms)                                                       time
                                                                                         time(ms) (ms)




          Panyi et al., Biophys. J., 1995, 69: 896-903;     Panyi and Deutsch, J.Gen.Physiol., 1996, 107:409-420.
          WT                        Mut

         m=0                         m=4                        m=1                  m=2                 m=3

                                               1.0




                          normalized current
                                                         intermediate inactivation kinetics
                                               0.5       (τ~30 ms)


                                                0
                                                     0             400               800
                                                                time (ms)

  •cooperative interaction between subunits
  •each subunit contributes equal free energy to inactivation
  •constraints the possible physical models of inactivation
Panyi et al., Biophys. J., 1995, 69: 896-903;                  Panyi and Deutsch, J.Gen.Physiol., 1996, 107:409-420.
    What kind of molecules inhibit
Kv1.3 and IKCa1 channels differentially?
                             peptide toxins block the pore
                 Tx


                 Tx




                  K++
                  K
                                     control
                                     wash


                                     toxin
200 pA




         10 ms
Péter et al., BBRC, 1998, 242:621-625;        Péter et al., BBRC, 2000, 278:34-37;
Péter et al, J. Membr.Biol., 2001, 179:13-25; Batista et al., Biochim. Biophys. Acta, 2002, 1601:123-131.
         Why is important to selectively block
                K+ channels of T cells?
        Naïve                TCM                           TEM



                   repeated antigen stimulus

IKCa1: 5-35/cell                                        ~50/cell

Kv1.3: ~200/cell                                      ~1500/cell




                                            -Multiple Sclerosis
                                            -Type-I (autoimmune) diabetes
                                            -Rheumatoid Arthritis

     proliferation of TEM in autoimmune diseases is selectively
                     inhibited by Kv1.3 blockers

                       Wulff et al., J.Clin.Invest., 2003, 111:1703-1713 (Chandy lab., UCLA)
                                                                               1.0




                                                fraction of blocked channels
                                                                               0.8

                                                                               0.6

                                                                               0.4

                                                                               0.2

                                                                               0.0




                                                                                       1.3

                                                                                                1
                                                                                                    1.2




                                                                                                                  R

                                                                                                                 .1
                                                                                                          ak .1
                                                                                             Ca




                                                                                                               -I

                                                                                                               v2
                                                                                                        Sh Kv1
                                                                                                    v
                                                                                        v
                                                                                            IK




                                                                                                             er

                                                                                                            rK
                                                                                                 hK
                                                                                     hK




                                                                                                           m
Anuroctoxin is a selective, high affinity blocker of Kv1.3


Bagdány et al., Mol. Pharmacol. 2005, 67:1034-1044; Olamendi-Portugal et al,., Toxicon, 2005, in press
           Can the microenvironment of the membrane
                  alter the channels’ behavior?


Does gating of Kv1.3 depend on the cholesterol content of the membrane?

Are Kv1.3 channels localized in specific microdomains of the membrane?




                                              CD4/CD8
                                  TCR/
                           raft   CD3

                                         p56lck
              Can cholesterol modulate channel kinetics?


                       1
normalized K current




                                                                                              1




                                                                       normalized K+current
                                         cholesterol loading

                                                                                                      control
+




                                              control
                                                                                                      cholesterol loading

                                                                                              0
                       0                                                                          0
                                                                                                  0             25          50
                           0              1                    2
                                       time (s)                                                           time (ms)



                               slower inactivation                                        slower and biphasic activation


                                               Hajdu et al., Pflugers Archives, 2003, 445:674-682.
Let’s see where the channels are!
   Kv1.3

T cell


 TCR/CD3




Kv1.3 and CD3 are highly co-localized

           Panyi et al., Proc. Natl. Acad. Sci. USA, 2003, 100:2592-2597.
Can we determine this relationship more precisely?



                                       hν’
            hν           FRET A
                     D             A
                         2-10 nm


                 D
Can we determine this relationship more precisely?




                                                                             0.06




                                              Relative frequency of pixels
   Kv1.3                                                                     0.05

                                                                             0.04
 T cell                                                                      0.03

                                                                             0.02

                                                                             0.01
 TCR/CD3
                                                                             0.00
                                                                                -30   -20    -10    0     10      20   30
                                                                                            FRET efficiency (%)




     Kv1.3 and TCR/CD3 are closely associated
           Panyi et al., Proc. Natl. Acad. Sci. USA, 2003, 100:2592-2597.
          Does this co-localization occur during a
              physiological immune response?

                 antigen presenting cell
            IS                TCR/CD3             LFA-1

T cell




 Structured recruitment of molecules in the immunological synapse
                                 Monks et al., Nature, 1998, 395:82-86 (Kupfer lab.)
     Is Kv1.3 also in the immune synapse?

                           Kv1.3             MHC I             CD8                    overlay

isolated T cell




                                                                                     JY



T cell attacking
CTL-JY conjugate
a target cell
                                                                              CTL                    CTL




                                                                                          JY




 CTL-JY conjugate                                                                   CTL

                                                                                     CTL




                                                                                               JY

                    Panyi et al, Proc. Natl. Acad. Sci. USA, 2004, 101:1285-1290.               JY
Are Kv1.3 channels in lipid rafts?


  Kv1.3                    lipid raft                  overlay




               Kv1.3 is localized to lipid rafts
                                                          CTL
          Panyi et al, Proc. Natl. Acad. Sci. USA, 2004, 101:1285-1290.
What is our model for the molecular interactions of Kv1.3
                in the immune synapse?


                                            FRET




                                                                   CD4/CD8
                              β1 integrin
                                                       TCR/




                                            Kv1.3
                                                       CD3

                                                              p56lck

                                                       hDlg
                                             Kvβ2   ZIP-1/2
                                                     PKC




    Panyi et al., Trends Immunol., 2004, 25: 565-569;          Panyi et al., Imm. Lett., 2004, 92:55-66.
               Mentors, co-workers and students




                           Carol Deutsch
                     U. of Pennsylavania, USA
Sándor Damjanovich

                                                  Lourival D. Possani
                                                   UNAM, Mexico



                                   Rezső Gáspár
            László Mátyus
                 Mentors, co-workers and students




                      Péter Hajdu
  Zoltán Varga
                                    Andrea Bodnár


                                                    Attila Jenei




                    György Vámosi
Miklós Bagdány                      Sándor Somodi

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:4
posted:8/9/2011
language:English
pages:26