Ionchannels and channelopaties in the heart

Viktória Szűts

Action of membrane transport protein

ATP-powered pump Ion chanels Transporters 101-103ions/s 107-108ions/s 102-104ions/s

• Cardiac K+ channels control the resting membrane potentials and the amplitude, duration, refractoriness and automaticity of action potentials. K+ channels share a similar structure, composed by four pore-forming α-subunits assembled as tetramers or dimers forming K+

selective pores and modulated by accessory subunits. The main K+channel pore forming protein is not translated from a single gene as Na+ and Ca+channels, but is made up of four separate subunits, which assembly with ß-subunits to form the functional channel More than 80 different K+ channels are expressed in the heart, display considerable diversity of native K+channels.

• Ca-independent transient outward potassium current (I to1) underlies by KCNA genes encoded Kv3.x and Kv4.x proteins.

• Delayed rectifier currents: the rapid (IKr) and slow (IKs) are encoded by different voltage-gated K+ channel genes. IKr is produced by the α-subunit ERG (KCNH2), in co-assemblance with the ß-subunit MiRP1 (KCNE2). IKs is produced by the α-subunit KvLQT1 (KCNQ) assembly with the accessories subunits of minK and MIPRs (KCNE1, KCNE2, KCNE3)

• Inward rectifier current (IK1) carried by Kir 2.1, Kir 2.2 and Kir 2.3 (KCNJ2, KCNJ12 and KCNJ4) channel proteins.

Nerbonne et al . Circ Res. 2001;89:944-956

Molecular composition of the cardiac K-ionchannelsSelectivity filter

Membrane topology of the Kv and Kir2.x K-ionchannels

H5 H5

Voltage gated K+channel Inward rectifier K+channel

Kv channel

CO2

CO2

CO2

Kv complex

NN

CC

KChAP PSD

MiRP

Gating movi

Ionchannels are open and close changing the permeability

Abott et al Neuropharm. 2004

Assembly of different ionchannel subunits

Intracellular

Extracellular

Molecular assembly of ion channels

Cavα Kvα Kir

Activation and Inactivation of The Sodium Channel

Sodium channels are characterized by voltage-dependent activation, rapid inactivation, and selective ion conductance. Depolarization of the cell membrane opens the ion pore allowing sodium to passively enter the cell down its concentration gradient . The increase in sodium conductance further depolarizes the membrane to near the sodium equilibrium potential. Inactivation of the sodium channel occurs within milliseconds, initiating a brief refractory period during which the membrane is not excitable. The mechanism of inactivation has been modeled as a "hinged lid" or "ball and chain", where the intracellular loop connecting domains III and IV of the a subunit closes the pore and prevents passage of sodium ions.

• Voltage-Gated Calcium Channels• Voltage-gated calcium channels are heteromultimers

composed of an α1 subunit and three auxiliary subunits, 2-δ, β and γ. The α1 subunit forms the ion pore and possesses gating functions and, in some cases, drug binding sites. Ten α1 subunits have been identified, which, in turn, are associated with the activities of the six classes of calcium channels. L-type channels have α1C (cardiac), α1D (neuronal/endocrine), α1S (skeletal muscle), and α1F (retinal) subunits; The α1 subunits each have four homologous domains (I-IV) that are composed of six transmembrane helices. The fourth transmembrane helix of each domain contains the voltage-sensing function. The four α1domains cluster in the membrane to form the ion pore. The β-subunit is localized intracellularly and is involved in the membrane trafficking of α1subunits. The γ-subunit is a glycoprotein having four transmembrane segments. The α2 subunit is a highly glycosylated extracellular protein that is attached to the membrane-spanning d-subunit by means of disulfide bonds. The α2-domain provides structural support required for channel stimulation, while the δ domain modulates the voltage-dependent activation and steady-state inactivation of the channel.

Abriel H. et al., Swiss Med Wkly 2004, 685-694. www.sm w. ch

Ionic currents and ion transporters responsible for cardiac action potential

• The expression and properties of these K+ channels are altered in cardiac diseases (ie. cardiac arrhythmia, Long QT syndrome, hypertrophyc cardiomyopathy, Andersen syndrome, heart failure). These K+ channels still require further investigation because they are involved in the basic normal heart rhythm, and may be altered in cardiac diseases.

Proposed cellular mechanism for the development of Torsade de pointes in the long QT syndrome

• Prolonged QT interval on ECG (reflects prolonged APD)• APD governed by a delicate balance between inward (Na+

or Ca+) and outward (K+) ionic current• Affecting the Na+ or Ca+ channel prolong APD via“gain-off-

function”mechanism, while mutation in genes encoding K+ channel by “loss-off-function” mechanism

Risk factors for developing Torsade de pointes

Abriel H. et al., Swiss Med Wkly 2004, 685-694.

Genetic variants (polymorphysm or mutations)

Ionic current, proteins and genes associated with inherited arrhythmias

Napolitano et al. Pharm. & ther. 2006,110:1-13

Congenital and aquired forms of long QT syndromes

Abriel H. et al., Swiss Med Wkly 2004, 685-694. www.sm w. ch

K+, Na+ channel LQT-associated genes and proteins

LQT3 Brugada Syndrome, Cardiac conduction defect, Sick sinus syndrome

SCN5AINa

LQT7 Andersen-Tawil Syndrome Kir2.1 (KCNJ2)Ik1

LQT8 Timothy Syndrome Cav1.2 (CACNA1c)ICaL

Kir6.2IkATP

Kir3.4IkAch

Progressziv familial heart Block1Kv1.7(KCNA7),Kv1.5Ikur

LQT2LQT6, FAF

HERG (KCNH2)MiRP1 (KCNE2)

IKr

LQT1, JLN1LQT5, JLN2

KvLQT1(KCNQ1)Mink (KCNE1)

IKs

LQTKv4.3ITo1

DiseaseGenesCurrent

AF

Gene mutations in LQT1 and LQT2

LQT1LQT2

HERGKCNH2

KvLQT1KCNQ1

Molecular structure and the membrane topology of the

HERG channel

Mutations in HERG channel

Atrial fibrillation (AF):

• Rapid shortening of the AERP• Functional changes of ion channel• Reduction of ICaL and gene expression of L-

type Ca channel• Increase in K+-ion channel activity of IkAch,

Ik1

• Reduction in Ito and Isus

• Reduced gene expression in Kv1.5, Kv4.3, Kir3.1, Kir3.4, Kir6.2

Pivotal role of Ser phosphorilation as a regulatory mechanism in Cav1.2 mode1/mode2 gating.

Timothy’s syndrome

ShortQT

HERG (KCNH2)Kir2.x (KCNJ2)

KvLQT1(KCNQ1)

IKr

IK1

IKs

Kv3.1, Kv3.4

DiseaseGenesCurrent

ICaCASQ2 (Calsequestrin2) CPVT

CPVT catecholamine-induced polymorphic ventricular tachycardia

RyR2 CPVT

β1-adrenoceptor (β1-AR)

β2-adrenoceptor (β2-AR)

Risk factor, modify disease orinfluence progression of disease

Risk factor, modify disease orinfluence progression of disease

AF

ICa

IkAch

Complexity of protein-protein interaction in cardiomyocytes

Missense mutation in calsequestrin2 (CASQ2)

Associated with autosomal recessive catecholamine-induced polymorphic ventricular tachycardia (CPVT)

SyncopeSeizures orSudden death

In response to Physical activity orEmotional stress

wild type

mutant

Kir2.1 ionchannel has an autosomal dominant mutation in Andersen-Tawil Syndrome

Cardiac arrhytmiasPeriodic paralysisDysmorphic bone structure(scoliosis,low-set ears, small chin, broad forehead

Facial and sceletal features

in Andersen-Tawil syndrome

Kir2.1 ion channel mutation

GIRK mutation

ANP role

• Gene-specific mutation study• Genexpression study• Microarray, qRT-PCR• Proteomica

kir2.x mRNA in dog & human

-0.002000000.000000000.002000000.004000000.00600000

0.008000000.010000000.012000000.01400000

Kir2.1 Kir2.2 Kir2.3 Kir2.4

HUMAN

DOG

Kir2.x analysisby RT-PCR

RV LV RA LA RV LV RA LADOG HUMAN

n=12 n= 6

0

1

2

3

4

5

6

HUMAN DOG

Rel

ativ

e am

ount

of K

v1.5

LV

LA

kDa7566

Expression of Kv1.5 protein in human and dog

Co-localization of Kv2 auxillary subunit with Kv1.5 in dog left ventricular myocytes

100

Kv1.5-FITCKv2-Texas red

Kv1.5-FITC Kv2-Texas red