pediatric cardiology

Ion Channels in the Cardiovascular System in Health and Disease

William A. Coetzeewac3@nyu.eduTel: 263-8518

Hearts are Composed of Cells

The Cardiac Myocyte

Cells Have Membranes

Channels

Pore

Filter

Gate

Patch Clamping

closed

open

Ion Channels - Gating

• A seminal contribution of Hodgkin and Huxley (circa 1940): channels transit among various conformational states

• Activation: process of channel opening during depolarization

• Inactivation: channels shut during maintained depolarization

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Inward Currents Outward Currents

+

+

K+

Na+Ca2+

Na+

K+

Ca2+

Cl-

Cl-

Cl-

Ion Channels

• Na+ channels

• Ca2+ channels

• K+ channels

• Exchangers

• Pumps

Na+ Channels - Electrophysiology

• Rapidly activating and inactivating

• A heart cell typically expresses more than 100,000 Na+ channels

• Responsible for the rapid upstroke of the cardiac action potential, and for rapid impulse conduction through cardiac tissue

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Ion Channels – The Traditional View of the Biophysicist

+

Ions move through “holes” in the membrane as a result of the electro-chemical driving force (flow of electrical current)

The “holes” are selective in that only certain ions are allowed to pass (i.e. Na+ or K+ or Ca2+, etc)

The “holes” or “channels” open and close randomly, but open kinetics are influenced by a) voltage and b) time

in

out

Ion Channels are Transmembrane Proteins

• The first molecular components of channels were identified only about a decade ago by molecular cloning methods

• The availability of channel cDNAs has allowed enormous progress in the understanding of the structure and molecular mechanisms of function of ion channels

• In addition to the pore forming or principal subunits (often called subunits), which determine the infrastructure of the channel, many channels (K+, Na+ and Ca2+ channels), contain auxiliary proteins that can modify the properties of the channels

Recent Advances

• Important new insights into the mechanisms of ionic selectivity, voltage- and calcium-dependent gating, inactivation and blockade of these channels have been obtained

• These efforts recently culminated with the crystallization and high resolution structural analysis of a K+ channel

The Na+ Channel -Subunit

Four repeating units.

Each domain folds into six transmembrane helices

Na+ Channels - Structure• Consist of various subunits,

but only the principal () subunit is required for function

• Four internally homologous domains (labeled I-IV)

• The four domains fold together so as to create a central pore

Marban et al, J Physiol (1998), 508.3, pp. 647-657

Na+ Channels:Structural elements of activation

• S4 segments serve as the activation sensors

• Charged residues in each S4 segment physically traverse the membrane

• Where are the activation gates?

Structural Elements of Gating and Selectivity

• Multiple inactivation processes exist

• Fast inactivation is mediated partly by the cytoplasmic linker between domains III and IV

• Slow inactivation?

Na+ Channels:Structural elements of inactivation

Principal and Auxiliary Subunits of Ion Channels

Na+-ChannelsModulation by auxiliary subunits

• Two distinct subunits (1 and 2) • Both contain:

– a small carboxy-terminal cytoplasmic domain, – a single membrane-spanning segment, and – a large amino-terminal extracellular domain with several consensus

sites for N-linked glycosylation and immunoglobulin-like folds

• The 1 subunit is widely expressed in skeletal muscle, heart and neuronal tissue, and is encoded by a single gene (SCN1B)

Na+-Channels: Genetic Disorders

• Congenital long-QT syndrome (LQT3)– Mutations in the

cardiac Na-channel gene (SCN5A)

– Slowed inactivation– Mutations reside at

loci consistent with this gating effect

Persistent inward current during AP repolarization, prolonging the QT interval and setting the stage for fatal ventricular arrhythmias

• Local anaesthetics (class I antiarrhythmic agents) block Na+ channels in a voltage-dependent manner (S6 segment of domain IV)

• Block is enhanced at depolarized potentials and/or with repetitive pulsing - modulated receptor model

• Neurotoxins: tetrodotoxin (TTX) interacts with a particular residue in the P region of domain I

• µ-conotoxins• Sea anemone (e.g. anthopleurin

A and B, ATX II) and scorpion toxins inhibit Na+ channel inactivation by binding to sites that include the S3-S4 extracellular loop of domain IV

Na+ Channels - Pharmacology

Ion Channels

• Na+ channels

• Ca2+ channels

• K+ channels

• Exchangers

• Pumps

Ca2+ Channels: Electrophysiology

• Calcium influx through voltage-dependent calcium channels triggers excitation-contraction coupling and regulates pacemaking activity in the heart.

• Multiple Ca2+ currents:– L, N, P, Q, R and T-type

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Two types of Ca2+ Currents in Heart

• L-type Ca2+ Current– High-voltage-activated– Slow inactivation (>500ms)– Large conductance (25pS)– DHP-sensitive– Requirement of

phosphorylation – Essential in triggering Ca2+

release from internal stores

• T-type Ca2+ Current– Low-voltage-activated– Low threshold of activation– Small conductance (8pS)– Slow activation & fast

inactivation– Slow deactivation!!– Blocked by mibefradil and

Ni2+ ions– Role in pacemaker activity?

The -subunit is known to contain the ion channel filter and has gating properties

The β-subunit is situated intracellularly and is involved in the membrane trafficking of α1-subunits.

The γ-subunit is a glycoprotein having four transmembrane segments.

The 2-subunit is a highly glycosylated extracellular protein that is attached to the membrane-spanning δ-subunit by means of disulfide bonds. The α2-subunit provides structural support whilst the δ-subunit modulates the voltage-dependent activation and steady-state inactivation of the channel

Ca2+ Channel -Subunits Molecular Composition

Gene Protein Type Chromosome Tissue

CACLN1A3 1S L-type 1q31-32 Skeletal

CACLN1A4 1A P/Q-type 19p13.1 Neuronal

CACLN1A5 1B N-type 9q34 Neuronal

CACLN1A1 1C L-type 12p13.3 Heart, VSM

CACLN1A2 1D L-type 3p14.3 Endocrine, brain

CACLN1A6 1E R-type? 1q25-31 Brain, heart

CACLNA1G 1G T-type 17q22 Brain, heart

CACLN1L21 2 7q21-22

CACLNB1 1 17q11.2-22

CACLNB1 17q23

Ca2+ Channel -Subunits Structural elements of function

Ca2+ Channel -Subunits Genetic Disorders

• Skeletal muscle • Mutations in CACNL1A3

(1S L-type skeletal muscle subunit)– Hypokalemic periodic

paralysis– Malignant hyperthermia

(mostly associated with RYR2)

• Neuronal• Mutations in CACNL1A4

(1A P/Q-type skeletal muscle subunit)– Familial hemiplegic

migraine– Episodic ataxia – Spinocerebellar ataxia

type-6

Skeletal Ca2+ Channel -Subunits Genetic Disorders

Hyperkalemic periodic paralysisMalignant hyperthermia

Ca2+ Channels: Pharmacology

• Three main classes of Ca2+ channel blockers:– Phenylalkylamines (verapamil)– Benzothiazipines (diltiazem)– Dihydropyridines (nifedipine)

• Bind to separate sites of the -subunit(common site: TMs 5&6 of repeat II and TM6 of repeat IV) – equivalent region in Na+ channel causes block by local anesthetics

Ion Channels

• Na+ channels

• Ca2+ channels

• K+ channels

• Exchangers

• Pumps

Functional Diversity of K+ Channels in the Heart

• Voltage-activated K+ Channels

• Inward rectifiers

• “Leak” K+ currents

Voltage-activated K+ Channels

K+

K+

+Voltage-activated

K+

-

Inward rectifierK+

“Leak”

Responsible for repolarization of the action potential and refractoriness (consequences for contractility and arrhythmias)

Inward Rectifier K+ Channels

K+

K+

+Voltage-activated

K+

-

Inward rectifierK+

“Leak”

Setting the resting potential and automaticity. Also responsible for repolarization of the action potential and refractoriness (consequences for contractility and arrhythmias)

Leak K+ Channels

• Plateau (IKP) K+ channels

K+

K+

+Voltage-activated

K+

-

Inward rectifierK+

“Leak”

“Leak” K+ channels:

Controlling action potential duration?

K+ Channels - Structure

• Both (principal) and (auxiliary) subunits exist

• Fortuitous correlation exists between the classification system based on function and that based on structure

K+ Channel Principal Subunits

Voltage-gated K+ channelsCa2+-activated K+ channels

“Leak” K+ channels Inward Rectifier K+ channels

6 TMD 4 TMD 2 TMD

Coetzee, 2001

K+ Channel Principal and Auxiliary SubunitsVoltage-gated K+ channelsCa2+-activated K+ channels

“Leak” K+ channels Inward Rectifier K+ channels

6 TMD 4 TMD 2 TMD

eag KCNQ SK slo Kv

eag erg elk

Kv1 Kv2 Kv3 Kv4 Kv5 Kv6 Kv8 Kv9

Kir1 Kir2 Kir3 Kir4 Kir5 Kir6 Kir7

KCNK1 KCNK9KCNK2 KCNK10KCNK3 KCNK12KCNK4 KCNK13KCNK5 KCNK15KCNK6 KCNK16KCNK7 KCNK17

Kir

SURKCR1minK

MiRPs KvKChAPKChIPs NCS1

Coetzee, 2001

Voltage-activated K+ Channels

• Transient outward current (Ito)

• Slowly activating delayed rectifier (IKs)

• Rapidly activating delayed rectifier (IKr)

• Ultra-rapidly activating delayed rectifier (IKur)

Responsible for repolarization of the action potential and refractoriness (consequences for contractility and arrhythmias)

Transient Outward K+ Channels

• Rapidly activating, slow inactivation

• Responsible for early repolarization (Purkinje fibers)

• Also contributes to late repolarization

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Compounds Blocking Ito

• Cations- TEA, Cs+, 4-AP

• Class I- Disopyramide- Quinidine- Flecainide - Propafenone

• Class III- Tedisamil

• Other- Caffeine, Ryanodine- Bepridil- D-600- Nifedipine- Imipramine

Delayed Rectifier Currents

IKr and IKs

Delayed Rectifier Current

Matsuura et al, 1987

Control Ca-free + Cd

Two Types of Delayed Rectifiers

Sanguinetti & Jurkiewicz, 1991

E-4031

550 ms

100 pA

Compounds Blocking Delayed Rectifiers

• Rapidly activating (IKr)- E-4031- Dofetilide - Sematilide- MK-499- La3+

• Slowly activating (IKs)- K+ sparing diuretics

• Indapamide • Triamterene

K+ Channel -Subunits Molecular determinants of gating

N-type inactivation

poreS4 segment

Kv Subunits Accelerate Inactivation of Kv Channels

Kv Subunits Increase Expression Levels of Kv Channels

Enhanced Surface Expression

Kv Subunits as Molecular Chaperones

3-Dimensional Structure of Kv2

Kv Confers Hypoxia-Sensitivity to Kv4 Channels

Identification of Frequenin as a Putative Kv4 -subunit

• We searched EST databases (using KChIP2 as a bait)

• Concentrated on ESTs cloned from cardiac libraries

• W81153: frequenin (cloned from a human fetal cardiac library)

Effects of Frequenin on Kv4.2 Currents

Kv4.2 + H2O Kv4.2 + Frequenin0

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Kv4.2 + Frequenin

Kv4.2+Frequenin

Kv4.2+H2O

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Frequenin Enhances Kv4.2 Membrane Trafficking

Kv4.2 Frequenin-GFP Kv4.2 + frequenin-GFP

Anti-Kv4.2 Ab Anti-Kv4.2 Ab

COS-7 cells

Delayed Rectifier K+ Channels Molecular Composition

• Rapidly-activating delayed rectifier– NCNH2 (h-erg)

• Slowly-activating delayed rectifier– KCNQ1 (KvLQT1) plus KCNE1 (minK)

• Ultra-rapidly activating delayed rectifier– Kv1.5?

Voltage-activated K+ Channels Pharmacology

• Transient outward current– 4-AP, bupivacaine, quinidine, profafenone, sotalol,

capsaicin, verapamil, nifedipine

• Rapidly-activating delayed rectifier– E-4031, dofetilide, sotalol, amiodarone, etc.

• Slowly-activating delayed rectifier– Quinidine, amiodarone, clofilium, indapamide

• Ultrarapid delayed rectifier– 4-AP, clofilium

Voltage-activated K+ Channels Genetic Disorders

Gene Channel Disease Chromosome

NCNA1 Kv1.1 Episodic Ataxia 12p13

NCNH2 H-erg LQT2 7q35-7q36

KCNQ1

KCNE1

KvLQT1

minK

LQT1 (Romano-Ward)

(Jervall-Lange-Nielsen)

11p15.5

21q22.1- 21q22.2

Mechanisms of Arrhythmias

• Abnormal automaticity

• Triggered activity

• Reentry

Triggered Activity

• Arrhythmias originating from afterdepolarizations– Early afterdepolarizations (phases 2 or 3)– Delayed afterdepolarizations (phase 4)

• If large enough, can engage Na+/Ca2+ channels and initiate an action potential

Early Afterdepolarizations

• Can occur when outward currents are inhibited or inward currents are enhanced

• Generally seen under conditions that prolong the action potential:– Hypokalemia, hypomagnesemia– Antiarrhythmic drugs

• Proposed mechanism for Torsades de Pointes

Factors Promoting EADs

• Autonomic - increased sympathetic tone- increased catecholamines- decreased parasympathetic

• Metabolic - hypoxia- acidosis

• Electrolytes - Cesium- Hypokalemia

Factors Promoting EADs

• Drugs - Sotalol- N-acetylprocainamide- Quinidine

• Heart rate - Bradycardia

Inward Rectifier K+ Channels

• The “classical” inward rectifier (IK1)

• G protein-activated K+ channels (IK,Ach; IK,Ado)

• ATP-sensitive K+ channels (IK,ATP)

• Na+-activated K+ channels

K+

K+

+Voltage-activated

K+

-

Inward rectifierK+

“Leak”

Inward rectifier K+ channels:

Setting the resting potential and automaticity. Also responsible for repolarization of the action potential and refractoriness (consequences for contractility and arrhythmias)

Inward Rectifier K+ ChannelsElectrophysiology

• Outward current under physiological conditions

• Less outward current when membrane is depolarized

• Open at all voltages

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Set the resting potential and automaticity. Also responsible for repolarization of the action potential and refractoriness (consequences for contractility and arrhythmias)

Inward Rectifier K+ ChannelsStructure

• Two transmembrane domains

• Pore• No voltage sensor

K+ Channel Principal Subunits

Voltage-gated K+ channelsCa2+-activated K+ channels

“Leak” K+ channels Inward Rectifier K+ channels

6 TMD 4 TMD 2 TMD

Coetzee, 2001

K+ Channel Principal and Auxiliary SubunitsVoltage-gated K+ channelsCa2+-activated K+ channels

“Leak” K+ channels Inward Rectifier K+ channels

6 TMD 4 TMD 2 TMD

eag KCNQ SK slo Kv

eag erg elk

Kv1 Kv2 Kv3 Kv4 Kv5 Kv6 Kv8 Kv9

Kir1 Kir2 Kir3 Kir4 Kir5 Kir6 Kir7

KCNK1 KCNK9KCNK2 KCNK10KCNK3 KCNK12KCNK4 KCNK13KCNK5 KCNK15KCNK6 KCNK16KCNK7 KCNK17

Kir

SURKCR1minK

MiRPs KvKChAPKChIPs NCS1

Coetzee, 2001

Inward Rectifier K+ ChannelsGenetic Disorders

Gene Channel Disease Chromosome

KCNJ1 Kir1.1 (ATP-activated K+ channel; renal)

Bartter’s syndrome 11q24

KCNJ2 Kir2.1 Anderson’s sydrome 17q23.1-q24.2

KCNJ8ABCC9

Kir6.1SUR2

Vasospastic angina??(Printzmetal’s angina)

12p11.2312p12.1

KCNJ11 Kir6.2 (ATP-sensitive K+ channel; pancreas)

Familial persistent hyperinsulinemic hypoglycemia of infancy

11p15.1

ABCC8 SUR1 Familial persistent hyperinsulinemic hypoglycemia of infancy

11p15.1

Inward Rectifier K+ ChannelsPharmacology

• “Classical” inward rectifiers– Ba2+, Cs+

• G protein-activated K+ channels– Acetylcholine, adenosine (mainly in atria)

• ATP-sensitive K+ channels– Blocked by glibenclamide– Opened by pinacidil, cromakalim, nicorandil

K+ Channel Principal and Auxiliary SubunitsVoltage-gated K+ channelsCa2+-activated K+ channels

“Leak” K+ channels Inward Rectifier K+ channels

6 TMD 4 TMD 2 TMD

eag KCNQ SK slo Kv

eag erg elk

Kv1 Kv2 Kv3 Kv4 Kv5 Kv6 Kv8 Kv9

Kir1 Kir2 Kir3 Kir4 Kir5 Kir6 Kir7

KCNK1 KCNK9KCNK2 KCNK10KCNK3 KCNK12KCNK4 KCNK13KCNK5 KCNK15KCNK6 KCNK16KCNK7 KCNK17

Kir

SURKCR1minK

MiRPs KvKChAPKChIPs NCS1

Coetzee, 2001

Role of the KATP Channel

Inagaki et al, 1995

Secretory Mechanisms

• Apocrine secretion occurs when the release of secretory materials is accompanied with loss of part of cytoplasm

• Holocrine secretion; the entire cell is secreted into the glandular lumen

• Exocytosis is the most commonly occurring type of secretion; here the secretory materials are contained in the secretory vesicles and released without loss of cytoplasm

Mechanism of Insulin Release

• Fasting state– Low cytosolic glucose– KATP channels are unblocked – High K+ conductance– Negative resting potential

-cellK+

• After a meal– Glucose taken up – Glycolysis

– KATP channels blocked

– Depolarization– Ca2+ influx– Secretory insulin release

stimulated

ATP

Glucose

Ca2+

Insulin

Mechanism of Insulin Release

Depolarization

Inward Rectifier K+ ChannelsGenetic Disorders

Gene Channel Disease Chromosome

KCNJ1 Kir1.1 (ATP-activated K+ channel; renal)

Bartter’s syndrome 11q24

KCNJ2 Kir2.1 Anderson’s sydrome 17q23.1-q24.2

KCNJ8ABCC9

Kir6.1SUR2

Vasospastic angina??(Printzmetal’s angina)

12p11.2312p12.1

KCNJ11 Kir6.2 (ATP-sensitive K+ channel; pancreas)

Familial persistent hyperinsulinemic hypoglycemia of infancy

11p15.1

ABCC8 SUR1 Familial persistent hyperinsulinemic hypoglycemia of infancy

11p15.1

Glibenclamide Blocks KATP Channels

Further Reading

• Frances M. Ashcroft. Ion Channels and Disease. Academic Press, 2000

• Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, Rudy B. Molecular diversity of K+ channels. Ann N Y Acad Sci 1999 Apr 30;868:233-85

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