The Heart Cardiac arrhythmia

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					            The Heart
                 Heart pumps 4300 gallons blood/day
                 100,000 heart beats/day y
                 35 million heart beats/year

RA               Missed beats/brief tachycardia not harmful



            LV

     RV




          Cardiac arrhythmia


                  Ventricular fibrillation causes
                  ~300,000 sudden deaths/year in U.S.

                  Most recurrent arrhythmias are acquired:
                  caused by M.I., heart failure, drugs

                   Inherited arrhythmias are rare:
                   most often caused by mutations
                   in ion channel genes




                                                              1
                                                      1 K currents
                                                               2
    Multiple currents                          INa             ICa
    mediate cardiac                             0                          3
                                                      INa = SCN5A
    action potentials                                                             4


                                                      Ito = Kv4.2, Kv4.3, Kv1.4
 Molecular basis of
 currents defined                              Ito                   IKs   IKs = KvLQT1/minK
                                                             IKur
 Most inherited
 arrhythmia caused by
 mutations in channel
 genes
                                                                                      Kir2 1
                                                                                IK1 = Kir2.1
                                                                               IK1
                                                        IKr = HERG/MiRP1
                                                                  I   Kr
                                              Ileak




        Inherited cardiac arrhythmias
Arrhythmia                              gene (muts) protein

Brugada syndrome (IVF)1                                  SCN5A (15)            Na channel
Isolated Cardiac Conduction                              SCN5A (4)             Na channel
       Disease2

Arrhythmogenic Right Ventricular                         RyR2 (4)              SR Ca
      Dysplasia3                                                               release
Catecholaminergic Polymorphic                            RyR2 (7)              channel
      Ventricular Tachycardia4

Wolf-Parkinson-White Syndrome5                           PRKAG2 (1)            kinase

1. Chen et al., Nature. 392: 293, 1998              4. Priori, et al., Circ 103:196, 2001
2. Tan et al., Nature. 409:1043                     5. Gollob et al, NEJM. 344:1823,2001
3. Tiso et al., Hum Mol Genet. 10:189, 2001




                                                                                               2
          Inherited cardiac arrhythmias
    Long QT Syndrome              locus             gene(s)          current

    Romano Ward
    Romano-Ward                   LQT3              SCN5A               INa
    (dominant)                    LQT2/6            HERG/KCNE2          IKr
                                  LQT1/5            KCNQ1/KCNE1         IKs
                                  LQT4                  ?

    Jervell & Lange-Nielsen                         KCNQ1/KCNE1         IKs
     (recessive, deafness)


    Andersen’s                    LQT7              KCNJ2               IK1
     (LQTS; bidirectional VT, periodic paralysis)




    Long QT syndrome
                                                    1. QTc interval > 450 ms
                                                    2. torsades de pointes
                                                            (syncope)
                                                    3. fibrillation
                                                     ……sudden death


                                       1

2




                             3




                                                                               3
               Early AfterDepolarizations:
        electrical trigger of Torsades de Pointes

                                             EAD
                                                                       py
                                                                  Therapy:
                                                                -adrenergic
                                                   ICaL           blockers




           NSR                             TdP




   Long QT syndrome: >220 mutations
Gene        encoded channel protein                       # of mutations
            rapid delayed rectifier (IKr)
HERG
   G                    subunit                              101
KCNE2             MiRP1  subunit                               4

            slow delayed rectifier (IKs)
KCNQ1             KVLQT1  subunit                              82
KCNE1             minK  subunit                                10

SCN5A       sodium channel (INa)  subunit                      10

KCNJ2       inward rectifier (IK1)  subunit                      15
          Gene Connection for the Heart http://pc4.fsm.it:81/cardmoc




                                                                               4
     Long QT syndrome: 3 examples

                         DIII DIV
• SCN5A (KPQ deletion in DIII-DIV linker)

• HERG (missense mutations in N-terminal
                 and pore helix domains)

• KCNE1 (missense mutations in C-terminus)




             Long QT syndrome
     SCN5A: mutations disrupt inactivation

         Wild-type Na channel                mutant (KPQ deletion)
                 currents                    Na channel currents
mV




                Bennett et al., Nature 376:683,1995




                                                                     5
QT interval = repolarization of ventricle
             Increase of sustained INa prolongs
                    APD and QT interval
                     INa
  +40 mV                        IKs
                                IKr
       INa                      IK1



-80 mV                                 0
                     200 msec
                 R

  P
                                              Q                               T
             Q   S
                                T




                                              1 K currents
                                                       2
      Multiple currents                INa              ICa
      mediate cardiac                   0                            3
                                              INa = SCN5A
      action potentials                                                   4


                                              Ito = Kv4.2, Kv4.3, Kv1.4
 Molecular basis of
 currents defined                      Ito                    IKs    IKs = KvLQT1/minK
                                                      IKur
 Most inherited
 arrhythmia caused by
 mutations in channel
 genes
                                                                                Kir2 1
                                                                          IK1 = Kir2.1
                                                                         IK1
                                                  IKr = HERG/MiRP1
                                                            I  Kr
                                      Ileak




                                                                                         6
    Long QT syndrome
    HERG: mutations in the pore helix
                                                                            T613M
                                                                                                                    V612L
                                                                   L615V
                  14Å


                                                                           Pore
                                                                           helix
                                                                                                          S5
                                                                                   S6

   A-gate



Zhou et al., (Nature 2001;411:657-661.).

                                                                                 S5-S6 of single HERG subunit




    HERG: pore helix mutations cause
                loss of channel
    function                                                      3.0

       W T HERG                                                   2.5     W T HERG
       (10 ng cRNA)
                                                                  2.0
                                                                  20         10 ng cRNA
                                                   current (A)




                                                                  1.5

                                                                  1.0

   2 A                                                           0.5                   5 ng cRNA

                                                                  0.0
               1s                                                   -80    -60   -40 -20       0     20   40   60
                                                                                   test potential (m V)




 0.5  A
              V612L                             T613M                                     L615V


                                              (30 ng cRNA)
                                           injected into oocytes




                                                                                                                            7
HERG: pore helix mutations disrupt trafficking of
         channels to plasma membrane

                                        40
                                        35
            Relati Light Units (x103)



                                                              Single oocyte chemiluminescence
                                        30                    of HA-tagged HERG subunits
                                        25
                                        20
                                        15
                 ive




                                        10
                                            5
                                            0
                                                 WT          Uninj.   V612L   T613M   L615V
                                                HERG                  HERG    HERG    HERG




      Increase of sustained INa or decrease in IK
            prolongs APD and QT interval

                                            INa
  +40 mV                                           IKs
                                                   IKr

      INa



-80 mV
                                            200 msec
                                        R

  P
                                                                         Q                    T
            Q                           S
                                                         T




                                                                                                  8
                                   1 K currents
                                            2
  Multiple currents         INa             ICa
  mediate cardiac            0                          3
                                   INa = SCN5A
  action potentials                                            4


                                   Ito = Kv4.2, Kv4.3, Kv1.4
Molecular basis of
currents defined            Ito                   IKs   IKs = KvLQT1/minK
                                          IKur
Most inherited
arrhythmia caused by
mutations in channel
genes
                                                                   Kir2 1
                                                             IK1 = Kir2.1
                                                            IK1
                                     IKr = HERG/MiRP1
                                               I   Kr
                           Ileak




KvLQT1 and minK subunits form IKs channels
  Mutations in either subunit cause LQTS


                  minK 
    KvLQT1 
          +
          +
          +   
          +




  IKs
channel



                                                         (CHO cells)




                                                                            9
                      KCNE1: long QT syndrome mutations

                                  H 2N
                                                           extracellular




                                                         minK subunit: 130 aa



                                                            intracellular
                               Ser74Leu
                                 Asp76Asn
                                                   COOH




KCNE1: mutations cause loss of function and/or shift
        in voltage dependence of IKs activation

 WT-IKs
                                                   D76N-IKs



                              WT-IKs
                 15           S74L-IKs
                              D76N-IKs


                 10
            A)
  current (A




                 5



                 0
                  -40   -20      0       20   40    60
                                     mV                  oocytes




                                                                                10
Summary: Inherited long QT syndrome

                       (            g
Mutations in SCN5A (Na channel gene):     )
     gain of function (disrupted inactivation)
     All Na channel mutations increase inward current

Mutations in KCNQ1, KCNE1, HERG, KCNE2, KCNJ2
(K channel subunit genes):
            ff        (   f                   ff      )
     loss of function (misfolding, altered trafficking)
     altered gating
     dominant-negative suppression
     All K channel mutations reduce outward current




                                                          11
 Ion channels in T cells, specific immunosuppression

1, What kind of cation channels can be found in T cells?
   How do their work?


2, Subtype specific expression of K+ channels in T cells


3, What kind of molecules inhibit K+ channels with
   high specificity and affinity?


4, Use of Kv1.3 inhibitors in in vivo experiments




  How do ion channels regulate the immune functions?

                                                          K+                     K+



                                            TCR/
                                                         Kv1.3




                                            CD3

                                                                 Em~-50 mV
                            IP3       PLC                                        CaM
              ER


                                                 Ca2+                                 proliferation
     CRAC
                                      [Ca2+]

                                               time

                                                      T cell


  Panyi et al., Trends Immunol., 2004, 25: 565-569;    Panyi et al., Imm. Lett., 2004, 92:55-66.




                                                                                                      12
                           How do K+ channels of T cells compare?
                                                                                  100
                                                                                  100
                                     +30 mV                                                       high [Ca2+]i




                                                                   urrent (pA)
               600
 urrent (pA)




                                     -10 mV                                        50
                                                                                   50




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




                                                                  cu
cu




                 0                                                               -150
                                                                                 -150

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




                Ion channels in T cells, specific immunosuppression

               1, What kind of cation channels can be found in T cells?
                  How do their work?


               2, Subtype specific expression of K+ channels in T cells


               3, What kind of molecules inhibit K+ channels with
                  high specificity and affinity?


               4, Use of Kv1.3 inhibitors in in vivo experiments




                                                                                                                        13
                        Kv1.3high TEM in MS, RA and T1DM

                  Naïve                       TCM                           TEM


                                   repeated antigen stimulus

                                                                        RA synovial fluid T cells
           Post-mortem MS
              brain slices
   CD3                     CD4+Kv1.3




                                                                      lls specific f i s li and GAD65
                                                               PB T cells s   ifi for insulin d

  Kv1.3+ cells          Kv1.3+ cells
  in parenchima         in perivascular
                        infiltrate


Beeton et al., PNAS, 2006, 103:17414-17419
Rus et al., PNAS, 2005, 102:11094-11099
 (Chandy lab., UCLA)




         Why Kv1.3 blockers are selective immunosuppressors?




                                                                                                    Kv1.3 blocker
                                                                                                    persistently
                                                                                                    inhibits
                                                                                                    proliferation




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




                                                                                                                    14
 Ion channels in T cells, specific immunosuppression

1, What kind of cation channels can be found in T cells?
   How do their work?


2, Subtype specific expression of K+ channels in T cells


3, What kind of molecules inhibit K+ channels with
   high specificity and affinity?


4, Use of Kv1.3 inhibitors in in vivo experiments




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




                      Panyi et al., Current Pharmaceutical Design, 2006, 12:2199-2220




                                                                                        15
                                                                             peptide toxins block the pore

                                                   Tx

                                                        x
                                                       Tx
                                                                                                                                 Lys27                             Tyr36


                                                                                                                                               dyad




                                                           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.




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

                                                 1.5
                                                                0.5 nM Anuroctoxin


                                                                                                                                 selectivity against IKCa1
                        peak current [nA]
                       peak current[nA]




                                                 1.0
                             c




                                                                                                                          2.5                    control
                                                 0.5
                                                                                                                          2.0
                                                                                                                                    Anuroctoxin (10 nM )
                                                                                                           current [nA]




                                                                                                                          1.5
                                                 0.0
                                                       0        200        400            600        800
                                                                                                                          1.0
                                                                           tim [s]
                                                                         time e [s]
                                                                                                                          0.5
                                                 1.0
                                   rent (I/I0)




                                                                                                                          0.0
                                                 0.8                                                                                                    IKCa1    Kv1.3 + IKCa1
          remaining fraction of curr




                                                                                                                          -0.5
                                                 0.6                                                                             -120 -100 -80    -60      -40   -20   0    20   40
                         RF




                                                                                                                                            m em brane potential [m V]
                                                 0.4
                                                            K d = 0.73 nM
                                                 0.2        n H = 0.99

                                                 0.0
                                                                0.1              1                  10
                                                                      Anuroctoxin [nM]
                                                                      Anuroctoxin [nM ]




                                                                                                                                                                                      16
    Anuroctoxin is a selective, high affinity blocker of Kv1.3

                                                Shaker IR                                                                                Kv2.1       wash-out
                                                                                control                                                              control
                                                                                wash-out                                                             Anuroctoxin
                                                                                A nuroctoxin                                                         (10 nM)
                                                                                (10 nM )




                                                                                                              2 nA
              3 nA




                                                           +50 m V                                                                +50 m V
                                                     -120 m V                                                                 -120 m V
                      10 m s                                                                                         10 ms




                                                     Kv1.1                                                                            Kv1.2
                                                                                                                                                 control
                                                                                                                                                 wash-out

                                                                           control




                                                                                                                                                               RF
                                                                           wash-out
                                                                           Anuroctoxin
                                                                           (10 nM)                                                               Anuroctoxin
                                                                                                                                                 (10 nM )
                                                                                                     600 pA
          200 pA




                                                                                                                             +50 mV
                                            +50 m V
                                         -120 mV                                                                       -120 mV

                     50 m s                                                                                   50 m s




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



                                                     1.0
                                           hannels




                                                     0.8
                      fraction of blocked ch




                                                     0.6

                                                     0.4

                                                     0.2

                                                     0.0
                                                                                           IR
                                                                      1




                                                                                    1




                                                                                                .1
                                                                 .3




                                                                            .2

                                                                                    1.
                                                                      Ca




                                                                                                v2
                                                                v1




                                                                           v1




                                                                                         r-
                                                                                 Kv
                                                                     IK
                                                            hK




                                                                          hK




                                                                                               rK
                                                                                      ke
                                                                                m
                                                                                    ha
                                                                                  S




Bagdány et al., Mol. Pharmacol. 2005, 67:1034-1044; Panyi et al., Curr. Pharm. Design, 2006, 12:2199-2220




                                                                                                                                                                    17
     Ion channels in T cells, specific immunosuppression

   1, What kind of cation channels can be found in T cells?
      How do their work?


   2, Subtype specific expression of K+ channels in T cells


   3, What kind of molecules inhibit K+ channels with
      high specificity and affinity?


   4, Use of Kv1.3 inhibitors in in vivo experiments




        Suppression of DTH reaction in rats by peptide toxins


               control                        treatment




DNFP = dinitrofluorobenzene




                                                                18
                                         0.8
                    ear thickness (mm)




                                         0.6
                                  (




                                         0.4

                                         0.2

                                         0.0
                                               10 μg   c
                                               toxin




                                         SUMMARY
1, Kv1.3, IKCa1 and CRAC channesl are expressed
   in T cells

2, Tem cells express exclusively Kv1.3, their proliferation
   can be selectively inhibited by Kv1.3 inhibitors

3, peptide toxins can be engineered to inhibit Kv1.3
   with high affinity and selectivity

4, Promising in vivo experiments point to the applicability
   of Kv1.3 inhibitors in autoimmune diseases




                                                              19

				
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