Ion Channels in the Cardiovascular System in Health and Disease
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Ion Channels in the Cardiovascular System in Health and Disease. William A. Coetzee [email protected] Tel: 263-8518. Hearts are Composed of Cells. The Cardiac Myocyte. Cells Have Membranes. Channels. Pore. Filter. Gate. Patch Clamping. open. closed. Ion Channels - Gating.

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Ion Channels in the Cardiovascular System in Health and Disease

William A. Coetzee

[email protected]

Tel: 263-8518





Channels l.jpg
Channels Disease

Pore

Filter

Gate



Slide9 l.jpg

open Disease

closed


Ion channels gating l.jpg
Ion Disease 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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+ Disease

+

Inward Currents

Outward Currents

Na+

K+

Ca2+

K+

Na+

Ca2+

Cl-

Cl-

Cl-


Ion channels l.jpg
Ion Channels Disease

  • Na+ channels

  • Ca2+ channels

  • K+ channels

  • Exchangers

  • Pumps


Na channels electrophysiology l.jpg
Na Disease+ 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


Ion channels the traditional view of the biophysicist l.jpg

+ Disease

Ion Channels – The Traditional View of the Biophysicist

out

in

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


Ion channels are transmembrane proteins l.jpg
Ion Channels are Transmembrane Proteins Disease

  • 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 a 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


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Recent Advances Disease

  • 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


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The Na Disease+ Channel a-Subunit

Four repeating units.

Each domain folds into six transmembrane helices


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Na Disease+ Channels - Structure

  • Consist of various subunits, but only the principal (a) 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


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Na Disease+ 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?



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Na Disease+ Channels:Structural elements of inactivation

  • Multiple inactivation processes exist

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

  • Slow inactivation?



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Na Disease+-ChannelsModulation by auxiliary subunits

  • Two distinct subunits (b1 and b2)

  • 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 b1 subunit is widely expressed in skeletal muscle, heart and neuronal tissue, and is encoded by a single gene (SCN1B)


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Na Disease+-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


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Local anaesthetics Disease (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


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Ion Channels Disease

  • Na+ channels

  • Ca2+ channels

  • K+ channels

  • Exchangers

  • Pumps


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Ca Disease2+ 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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L-type Ca Disease2+ 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?

Two types of Ca2+ Currents in Heart


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The Diseasea1-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 a2-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


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Ca Disease2+ Channel a-Subunits Molecular Composition


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Ca Disease2+ Channel a-Subunits Structural elements of function


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Skeletal muscle Disease

Mutations in CACNL1A3 (a1S L-type skeletal muscle subunit)

Hypokalemic periodic paralysis

Malignant hyperthermia (mostly associated with RYR2)

Neuronal

Mutations in CACNL1A4 (a1A P/Q-type skeletal muscle subunit)

Familial hemiplegic migraine

Episodic ataxia

Spinocerebellar ataxia type-6

Ca2+ Channel a-Subunits Genetic Disorders


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Skeletal Ca Disease2+ Channel a-Subunits Genetic Disorders

Hyperkalemic periodic paralysis

Malignant hyperthermia


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Ca Disease2+ Channels: Pharmacology

  • Three main classes of Ca2+ channel blockers:

    • Phenylalkylamines (verapamil)

    • Benzothiazipines (diltiazem)

    • Dihydropyridines (nifedipine)

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


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Ion Channels Disease

  • Na+ channels

  • Ca2+ channels

  • K+ channels

  • Exchangers

  • Pumps


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Functional Diversity of K Disease+ Channels in the Heart

  • Voltage-activated K+ Channels

  • Inward rectifiers

  • “Leak” K+ currents


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Voltage-activated Disease

K+

+

K+

-

“Leak”

K+

K+

Inward rectifier

Voltage-activated K+ Channels

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


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Voltage-activated Disease

K+

+

K+

-

“Leak”

K+

K+

Inward rectifier

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)


Slide41 l.jpg

Voltage-activated Disease

K+

+

K+

-

“Leak”

K+

K+

Inward rectifier

Leak K+ Channels

“Leak” K+ channels:

  • Plateau (IKP) K+ channels

Controlling action potential duration?


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K Disease+ Channels - Structure

  • Both a (principal) and b (auxiliary) subunits exist

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


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K Disease+ Channel Principal Subunits

Voltage-gated K+ channels

Ca2+-activated K+ channels

“Leak” K+ channels

Inward Rectifier

K+ channels

6 TMD

4 TMD

2 TMD

Coetzee, 2001


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K Disease+ Channel Principal and Auxiliary Subunits

Voltage-gated K+ channels

Ca2+-activated K+ channels

“Leak” K+ channels

Inward Rectifier

K+ channels

6 TMD

4 TMD

2 TMD

KCR1

minK

MiRPs

KCNK1 KCNK9

KCNK2 KCNK10

KCNK3 KCNK12

KCNK4 KCNK13

KCNK5 KCNK15

KCNK6 KCNK16

KCNK7 KCNK17

SUR

Kvb

KChAP

KChIPs

NCS1

Kir

eag

KCNQ

SK

slo

Kv

Kir1

Kir2

Kir3

Kir4

Kir5

Kir6

Kir7

eag

erg

elk

Kv1

Kv2

Kv3

Kv4

Kv5

Kv6

Kv8

Kv9

Coetzee, 2001


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Voltage-activated K Disease+ 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)


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Transient Outward K Disease+ Channels

  • Rapidly activating, slow inactivation

  • Responsible for early repolarization (Purkinje fibers)

  • Also contributes to late repolarization


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Cations Disease

TEA, Cs+, 4-AP

Class I

Disopyramide

Quinidine

Flecainide

Propafenone

Class III

Tedisamil

Other

Caffeine, Ryanodine

Bepridil

D-600

Nifedipine

Imipramine

Compounds Blocking Ito


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Delayed Rectifier Currents Disease

IKr and IKs


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Delayed Rectifier Current Disease

Control

Ca-free + Cd

Matsuura et al, 1987


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Two Types of Delayed Rectifiers Disease

550 ms

E-4031

100 pA

Sanguinetti & Jurkiewicz, 1991


Compounds blocking delayed rectifiers l.jpg

Rapidly activating (I DiseaseKr)

E-4031

Dofetilide

Sematilide

MK-499

La3+

Slowly activating (IKs)

K+ sparing diuretics

Indapamide

Triamterene

Compounds Blocking Delayed Rectifiers


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K Disease+ Channel a-Subunits Molecular determinants of gating

pore

S4 segment

N-type inactivation


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Kv Diseaseb Subunits Accelerate Inactivation of Kv Channels


Slide54 l.jpg

Kv Diseaseb Subunits Increase Expression Levels of Kv Channels



Slide56 l.jpg

Kv Diseaseb Subunits as Molecular Chaperones



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Kv Diseaseb Confers Hypoxia-Sensitivity to Kv4 Channels


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Identification of Frequenin as a Putative Kv4 Diseaseb-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)


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Kv4.2+H Disease2O

Kv4.2+Frequenin

10 mA

100 ms

Effects of Frequenin on Kv4.2 Currents

*

20

15

10

5

0

Kv4.2 + H2O

Kv4.2 + Frequenin


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Frequenin Enhances Kv4.2 Membrane Trafficking Disease

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 l.jpg
Delayed Rectifier K Disease+ ChannelsMolecular 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 l.jpg
Voltage-activated K Disease+ 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


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Voltage-activated K Disease+ Channels Genetic Disorders


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Mechanisms of Arrhythmias Disease

  • Abnormal automaticity

  • Triggered activity

  • Reentry


Triggered activity l.jpg
Triggered Activity Disease

  • 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 l.jpg
Early Afterdepolarizations Disease

  • 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 l.jpg

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

Metabolic - hypoxia - acidosis

Electrolytes - Cesium - Hypokalemia

Factors Promoting EADs


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Factors Promoting EADs Disease

  • Drugs - Sotalol - N-acetylprocainamide - Quinidine

  • Heart rate - Bradycardia


Slide72 l.jpg

Voltage-activated Disease

K+

+

K+

-

“Leak”

K+

K+

Inward rectifier

Inward Rectifier K+ Channels

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

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


Inward rectifier k channels electrophysiology l.jpg
Inward Rectifier K Disease+ ChannelsElectrophysiology

  • Outward current under physiological conditions

  • Less outward current when membrane is depolarized

  • Open at all voltages

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


Inward rectifier k channels structure l.jpg
Inward Rectifier K Disease+ ChannelsStructure

  • Two transmembrane domains

  • Pore

  • No voltage sensor


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K Disease+ Channel Principal Subunits

Voltage-gated K+ channels

Ca2+-activated K+ channels

“Leak” K+ channels

Inward Rectifier

K+ channels

6 TMD

4 TMD

2 TMD

Coetzee, 2001


Slide76 l.jpg

K Disease+ Channel Principal and Auxiliary Subunits

Voltage-gated K+ channels

Ca2+-activated K+ channels

“Leak” K+ channels

Inward Rectifier

K+ channels

6 TMD

4 TMD

2 TMD

KCR1

minK

MiRPs

KCNK1 KCNK9

KCNK2 KCNK10

KCNK3 KCNK12

KCNK4 KCNK13

KCNK5 KCNK15

KCNK6 KCNK16

KCNK7 KCNK17

SUR

Kvb

KChAP

KChIPs

NCS1

Kir

eag

KCNQ

SK

slo

Kv

Kir1

Kir2

Kir3

Kir4

Kir5

Kir6

Kir7

eag

erg

elk

Kv1

Kv2

Kv3

Kv4

Kv5

Kv6

Kv8

Kv9

Coetzee, 2001


Inward rectifier k channels genetic disorders l.jpg
Inward Rectifier K Disease+ ChannelsGenetic Disorders


Inward rectifier k channels pharmacology l.jpg
Inward Rectifier K Disease+ 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


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K Disease+ Channel Principal and Auxiliary Subunits

Voltage-gated K+ channels

Ca2+-activated K+ channels

“Leak” K+ channels

Inward Rectifier

K+ channels

6 TMD

4 TMD

2 TMD

KCR1

minK

MiRPs

KCNK1 KCNK9

KCNK2 KCNK10

KCNK3 KCNK12

KCNK4 KCNK13

KCNK5 KCNK15

KCNK6 KCNK16

KCNK7 KCNK17

SUR

Kvb

KChAP

KChIPs

NCS1

Kir

eag

KCNQ

SK

slo

Kv

Kir1

Kir2

Kir3

Kir4

Kir5

Kir6

Kir7

eag

erg

elk

Kv1

Kv2

Kv3

Kv4

Kv5

Kv6

Kv8

Kv9

Coetzee, 2001


Slide80 l.jpg

Role of the K DiseaseATP Channel

  • Inagaki et al, 1995


Secretory mechanisms l.jpg
Secretory Mechanisms Disease

  • 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


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Mechanism of Insulin Release Disease

  • Fasting state

    • Low cytosolic glucose

    • KATP channels are unblocked

    • High K+ conductance

    • Negative resting potential

b-cell

K+


Mechanism of insulin release83 l.jpg

After a meal Disease

Glucose taken up

Glycolysis

KATP channels blocked

Depolarization

Ca2+ influx

Secretory insulin release stimulated

Mechanism of Insulin Release

Glucose

Insulin

ATP

Ca2+

Depolarization


Inward rectifier k channels genetic disorders84 l.jpg
Inward Rectifier K Disease+ ChannelsGenetic Disorders


Slide86 l.jpg

Glibenclamide Blocks K DiseaseATP Channels


Further reading l.jpg
Further Reading Disease

  • 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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Next Thursday Disease


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