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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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channels
Channels

Pore

Filter

Gate

slide9

open

closed

ion channels gating
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
slide12

+

+

Inward Currents

Outward Currents

Na+

K+

Ca2+

K+

Na+

Ca2+

Cl-

Cl-

Cl-

ion channels
Ion Channels
  • Na+ channels
  • Ca2+ channels
  • K+ channels
  • Exchangers
  • Pumps
na channels electrophysiology
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
ion channels the traditional view of the biophysicist

+

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
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 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
recent advances
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 a subunit
The Na+ Channel a-Subunit

Four repeating units.

Each domain folds into six transmembrane helices

na channels structure
Na+ 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

na channels structural elements of activation
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?
na channels structural elements of inactivation
Na+ 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?
na channels modulation by auxiliary subunits
Na+-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)
na channels genetic disorders
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

na channels pharmacology
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 channels28
Ion Channels
  • Na+ channels
  • Ca2+ channels
  • K+ channels
  • Exchangers
  • Pumps
ca 2 channels electrophysiology
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
two types of ca 2 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?

Two types of Ca2+ Currents in Heart
slide31

The a1-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

ca 2 channel a subunits genetic disorders
Skeletal muscle

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
skeletal ca 2 channel a subunits genetic disorders
Skeletal Ca2+ Channel a-Subunits Genetic Disorders

Hyperkalemic periodic paralysis

Malignant hyperthermia

ca 2 channels pharmacology
Ca2+ 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
ion channels37
Ion Channels
  • Na+ channels
  • Ca2+ channels
  • K+ channels
  • Exchangers
  • Pumps
functional diversity of k channels in the heart
Functional Diversity of K+ Channels in the Heart
  • Voltage-activated K+ Channels
  • Inward rectifiers
  • “Leak” K+ currents
voltage activated k channels

Voltage-activated

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)

slide40

Voltage-activated

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

Voltage-activated

K+

+

K+

-

“Leak”

K+

K+

Inward rectifier

Leak K+ Channels

“Leak” K+ channels:

  • Plateau (IKP) K+ channels

Controlling action potential duration?

k channels structure
K+ 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
slide43

K+ 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

slide44

K+ 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

voltage activated k channels45
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
Transient Outward K+ Channels
  • Rapidly activating, slow inactivation
  • Responsible for early repolarization (Purkinje fibers)
  • Also contributes to late repolarization
compounds blocking i to
Cations

TEA, Cs+, 4-AP

Class I

Disopyramide

Quinidine

Flecainide

Propafenone

Class III

Tedisamil

Other

Caffeine, Ryanodine

Bepridil

D-600

Nifedipine

Imipramine

Compounds Blocking Ito
delayed rectifier current
Delayed Rectifier Current

Control

Ca-free + Cd

Matsuura et al, 1987

two types of delayed rectifiers
Two Types of Delayed Rectifiers

550 ms

E-4031

100 pA

Sanguinetti & Jurkiewicz, 1991

compounds blocking delayed rectifiers
Rapidly activating (IKr)

E-4031

Dofetilide

Sematilide

MK-499

La3+

Slowly activating (IKs)

K+ sparing diuretics

Indapamide

Triamterene

Compounds Blocking Delayed Rectifiers
identification of frequenin as a putative kv4 b subunit
Identification of Frequenin as a Putative Kv4 b-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)
slide60

Kv4.2+H2O

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

frequenin enhances kv4 2 membrane trafficking
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
Delayed Rectifier K+ 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
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
mechanisms of arrhythmias
Mechanisms of Arrhythmias
  • Abnormal automaticity
  • Triggered activity
  • Reentry
triggered activity
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
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
factors promoting eads71
Factors Promoting EADs
  • Drugs - Sotalol - N-acetylprocainamide - Quinidine
  • Heart rate - Bradycardia
slide72

Voltage-activated

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
Inward Rectifier K+ 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
Inward Rectifier K+ ChannelsStructure
  • Two transmembrane domains
  • Pore
  • No voltage sensor
slide75

K+ 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

K+ 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 pharmacology
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
slide79

K+ 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

Role of the KATP Channel

  • Inagaki et al, 1995
secretory mechanisms
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
Mechanism of Insulin Release
  • Fasting state
    • Low cytosolic glucose
    • KATP channels are unblocked
    • High K+ conductance
    • Negative resting potential

b-cell

K+

mechanism of insulin release83
After a meal

Glucose taken up

Glycolysis

KATP channels blocked

Depolarization

Ca2+ influx

Secretory insulin release stimulated

Mechanism of Insulin Release

Glucose

Insulin

ATP

Ca2+

Depolarization

further reading
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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