The Heart Heart pumps 4300 gallons blood/day 100,000 heart beats/dayy 35 million heart beats/year Missed beats/brief tachycardia not harmful

RA

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

INa

Multiple currents mediate cardiac action potentials

0

ICa 3

INa = SCN5A

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

IKr = HERG/MiRP1 I

IK1 = Kir2.1 Kir2 1 IK1

Kr

Ileak

Inherited cardiac arrhythmias Arrhythmia

gene (muts) protein

Brugada syndrome (IVF)1 Isolated Cardiac Conduction Disease2

SCN5A (15) SCN5A (4)

Na channel Na channel

Arrhythmogenic Right Ventricular Dysplasia3 Catecholaminergic Polymorphic Ventricular Tachycardia4

RyR2 (4) RyR2 (7)

SR Ca release channel

Wolf-Parkinson-White Syndrome5

PRKAG2 (1)

kinase

1. Chen et al., Nature. 392: 293, 1998 2. Tan et al., Nature. 409:1043 3. Tiso et al., Hum Mol Genet. 10:189, 2001

4. Priori, et al., Circ 103:196, 2001 5. Gollob et al, NEJM. 344:1823,2001

2

Inherited cardiac arrhythmias Long QT Syndrome

locus

gene(s)

Romano-Ward Romano Ward

LQT3 LQT2/6 LQT1/5 LQT4

SCN5A HERG/KCNE2 KCNQ1/KCNE1 ?

INa IKr IKs

KCNQ1/KCNE1

IKs

KCNJ2

IK1

(dominant)

Jervell & Lange-Nielsen

current

(recessive, deafness)

Andersen’s

LQT7

(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

Therapy: py -adrenergic blockers

ICaL

NSR

TdP

Long QT syndrome: >220 mutations Gene HERG G KCNE2

encoded channel protein rapid delayed rectifier (IKr)  subunit MiRP1  subunit

KCNQ1 KCNE1

slow delayed rectifier (IKs) KVLQT1  subunit minK  subunit

82 10

SCN5A

sodium channel (INa)  subunit

10

KCNJ2

# of mutations 101 4

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

4

Long QT syndrome: 3 examples • SCN5A (KPQ deletion in DIII-DIV 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 currents

mutant (KPQ deletion) 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 IK1

INa

-80 mV

0

200 msec R

P

Q

S

Q

T

T

1 K currents 2

Multiple currents mediate cardiac action potentials

Molecular basis of currents defined

INa 0

ICa 3

INa = SCN5A

4

Ito = Kv4.2, Kv4.3, Kv1.4 Ito

IKs

IKs = KvLQT1/minK

IKur

Most inherited arrhythmia caused by mutations in channel genes

IKr = HERG/MiRP1 I

IK1 = Kir2.1 Kir2 1 IK1

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 (10 ng cRNA)

2.5

W T HERG

current (A)

20 2.0

2 A

10 ng cRNA

1.5 1.0 0.5

5 ng cRNA

0.0 1s

0.5  A

V612L

-80

-60

T613M

-40 -20 0 20 test potential (m V)

40

60

L615V

(30 ng cRNA) injected into oocytes

7

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

Relatiive Light Units (x103)

40 35

Single oocyte chemiluminescence of HA-tagged HERG subunits

30 25 20 15 10 5 0

WT HERG

Uninj.

V612L HERG

T613M HERG

L615V 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

S

T

Q

T

8

1 K currents 2

Multiple currents mediate cardiac action potentials

Molecular basis of currents defined

INa 0

ICa 3

INa = SCN5A

4

Ito = Kv4.2, Kv4.3, Kv1.4 Ito

IKs

IKs = KvLQT1/minK

IKur

Most inherited arrhythmia caused by mutations in channel genes

IKr = HERG/MiRP1 I

IK1 = Kir2.1 Kir2 1 IK1

Kr

Ileak

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

KvLQT1  + + + +

minK 



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 S74L-IKs D76N-IKs

current (A A)

15

10

5

0 -40

-20

0

20 mV

40

60

oocytes

10

Summary: Inherited long QT syndrome Mutations in SCN5A ((Na channel gene): g ) gain of function (disrupted inactivation) All Na channel mutations increase inward current Mutations in KCNQ1, KCNE1, HERG, KCNE2, KCNJ2 (K channel subunit genes): loss off function f (misfolding, ( f altered trafficking) ff ) 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+

ER

IP3

Kv1.3

TCR/ CD3

Em~-50 mV

PLC

Ca2+ CRAC

CaM

proliferation

[Ca2+] time

T cell

Panyi et al., Trends Immunol., 2004, 25: 565-569;

Panyi et al., Imm. Lett., 2004, 92:55-66.

12

600

+30 mV

400

-50 mV -80 mV

100 100

cu urrent (pA) cur rent (pA)

cu urrent (pA)

How do K+ channels of T cells compare? -10 mV

200 0 0

200

400

600

800

time (ms)

Kv1.3 Gating: Single channel conductance

voltage-gated similar (~10 pS)

1000

high [Ca2+]i

50 50 0

low [Ca2+]i

-50 -50 -100 100 100 -100 -150 -150 -140 -140 -120 -120 -100 -100 -80 -80

-60 -60

-40 -40

membrane potential(mV) (mV) membrane potential

IKCa1 Ca2+-activated similar (~10 pS)

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 CD4+Kv1.3

CD3

PB T cells lls sspecific ifi f for insulin i s li and d GAD65 Kv1.3+ cells in parenchima

Kv1.3+ cells 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 Tx x Tyr36

Lys27 dyad

K++ control wash

200 pA

toxin

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

peakcccurrent peak urrent[nA] [nA]

0.5 nM Anuroctoxin

selectivity against IKCa1

1.0

2.5

control

0.5

2.0

0

200

400

600

800

remaining fraction RF of currrent (I/I0)

time tim e [s][s]

current [nA]

Anuroctoxin (10 nM ) 0.0

1.5 1.0 0.5

1.0

0.0

0.8

IKCa1

Kv1.3 + IKCa1

-0.5 0.6 0.4 0.2

-120 -100 -80

-60

-40

-20

0

20

40

m em brane potential [m V]

K d = 0.73 nM n H = 0.99

0.0 0.1

1 Anuroctoxin [nM]] Anuroctoxin [nM

10

16

Anuroctoxin is a selective, high affinity blocker of Kv1.3 Kv2.1

Shaker IR control wash-out

2 nA

3 nA

A nuroctoxin (10 nM )

+50 m V 10 m s

+50 m V -120 m V

-120 m V

10 ms

Kv1.2

Kv1.1

control wash-out RF

control wash-out Anuroctoxin (10 nM)

Anuroctoxin (10 nM )

600 pA

200 pA

wash-out control Anuroctoxin (10 nM)

+50 m V -120 mV

+50 mV -120 mV

50 m s

50 m s

1.0 0.8 0.6 0.4 0.2

.1 v2

IR

rK

1 1. S

ha

ke

r-

.2

Kv m

v1

Ca

hK

v1

IK

hK

1

0.0

.3

fraction of blocked ch hannels

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

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

ear thickness ((mm)

0.8 0.6 0.4 0.2 0.0

10 μg toxin

c

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