pediatric cardiology
TRANSCRIPT
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Ion Channels in the Cardiovascular System in Health and Disease
William A. [email protected]: 263-8518
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Hearts are Composed of Cells
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The Cardiac Myocyte
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Cells Have Membranes
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Channels
Pore
Filter
Gate
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Patch Clamping
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closed
open
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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
0 500 1000
0
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Inward Currents Outward Currents
+
+
K+
Na+Ca2+
Na+
K+
Ca2+
Cl-
Cl-
Cl-
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Ion Channels
• Na+ channels
• Ca2+ channels
• K+ channels
• Exchangers
• Pumps
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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
0 100 200 300 400 500-45
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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
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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
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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
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The Na+ Channel -Subunit
Four repeating units.
Each domain folds into six transmembrane helices
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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
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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?
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Structural Elements of Gating and Selectivity
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• 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
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Principal and Auxiliary Subunits of Ion Channels
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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)
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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
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• 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
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Ion Channels
• Na+ channels
• Ca2+ channels
• K+ channels
• Exchangers
• Pumps
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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
0 100 200 300 400 500 600
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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?
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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
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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
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Ca2+ Channel -Subunits Structural elements of function
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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
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Skeletal Ca2+ Channel -Subunits Genetic Disorders
Hyperkalemic periodic paralysisMalignant hyperthermia
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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
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Ion Channels
• Na+ channels
• Ca2+ channels
• K+ channels
• Exchangers
• Pumps
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Functional Diversity of K+ Channels in the Heart
• Voltage-activated K+ Channels
• Inward rectifiers
• “Leak” K+ currents
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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)
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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)
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Leak K+ Channels
• Plateau (IKP) K+ channels
K+
K+
+Voltage-activated
K+
-
Inward rectifierK+
“Leak”
“Leak” K+ channels:
Controlling action potential duration?
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K+ Channels - Structure
• Both (principal) and (auxiliary) subunits exist
• Fortuitous correlation exists between the classification system based on function and that based on structure
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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
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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
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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)
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Transient Outward K+ Channels
• Rapidly activating, slow inactivation
• Responsible for early repolarization (Purkinje fibers)
• Also contributes to late repolarization
0 100 200 300 400 500-0.2
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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
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Delayed Rectifier Currents
IKr and IKs
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Delayed Rectifier Current
Matsuura et al, 1987
Control Ca-free + Cd
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Two Types of Delayed Rectifiers
Sanguinetti & Jurkiewicz, 1991
E-4031
550 ms
100 pA
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Compounds Blocking Delayed Rectifiers
• Rapidly activating (IKr)- E-4031- Dofetilide - Sematilide- MK-499- La3+
• Slowly activating (IKs)- K+ sparing diuretics
• Indapamide • Triamterene
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K+ Channel -Subunits Molecular determinants of gating
N-type inactivation
poreS4 segment
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Kv Subunits Accelerate Inactivation of Kv Channels
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Kv Subunits Increase Expression Levels of Kv Channels
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Enhanced Surface Expression
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Kv Subunits as Molecular Chaperones
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3-Dimensional Structure of Kv2
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Kv Confers Hypoxia-Sensitivity to Kv4 Channels
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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)
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Effects of Frequenin on Kv4.2 Currents
Kv4.2 + H2O Kv4.2 + Frequenin0
5
10
15
20 *
-100 -80 -60 -40 -20 20 40 60
10
20
A
Kv4.2 + H2O
Kv4.2 + Frequenin
mV
0.0
0.5
1.0
200 ms
Nor
mal
ized
Cur
rent
s
Kv4.2+H2O
Kv4.2 + Frequenin
Kv4.2+Frequenin
Kv4.2+H2O
100 ms
10 A
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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
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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?
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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
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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
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Mechanisms of Arrhythmias
• Abnormal automaticity
• Triggered activity
• Reentry
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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
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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
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Factors Promoting EADs
• Autonomic - increased sympathetic tone- increased catecholamines- decreased parasympathetic
• Metabolic - hypoxia- acidosis
• Electrolytes - Cesium- Hypokalemia
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Factors Promoting EADs
• Drugs - Sotalol- N-acetylprocainamide- Quinidine
• Heart rate - Bradycardia
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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)
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Inward Rectifier K+ ChannelsElectrophysiology
• Outward current under physiological conditions
• Less outward current when membrane is depolarized
• Open at all voltages
0 100 200 300 400 500
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Inw
ard
Re
ctifie
r C
urr
en
t (
A)
time
0 100 200 300 400 500
-80
-40
0
40
mV
Set the resting potential and automaticity. Also responsible for repolarization of the action potential and refractoriness (consequences for contractility and arrhythmias)
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Inward Rectifier K+ ChannelsStructure
• Two transmembrane domains
• Pore• No voltage sensor
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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
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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
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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
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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
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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
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Role of the KATP Channel
Inagaki et al, 1995
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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
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Mechanism of Insulin Release
• Fasting state– Low cytosolic glucose– KATP channels are unblocked – High K+ conductance– Negative resting potential
-cellK+
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• 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
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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
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Glibenclamide Blocks KATP Channels
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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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Next Thursday