Coetzee-IONCHANN

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Information about Coetzee-IONCHANN
Science-Technology

Published on January 12, 2009

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Slide 1: Ion Channels in the Cardiovascular System in Health and Disease William A. Coetzee wac3@nyu.edu Tel: 263-8518 Hearts are Composed of Cells : Hearts are Composed of Cells The Cardiac Myocyte : The Cardiac Myocyte Cells Have Membranes : Cells Have Membranes Channels : Channels Pore Filter Gate Patch Clamping : Patch Clamping Slide 9: closed open 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 Slide 12: Inward Currents Outward Currents K+ Na+ Ca2+ Na+ K+ 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 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 : 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? Structural Elements of Gating and Selectivity : Structural Elements of Gating and Selectivity 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:Structural elements of inactivation Slide 24: Principal and Auxiliary Subunits of Ion Channels Na+-ChannelsModulation 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 Channels : Ion Channels Na+ channels Ca2+ channels K+ channels Exchangers Pumps Ca2+ 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 Ca2+ Currents in Heart : 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? Slide 31: The a1-subunit is known to contain the ion channel filter and has gating properties The ß-subunit is situated intracellularly and is involved in the membrane trafficking of a1-subunits. The ?-subunit is a glycoprotein having four transmembrane segments. The a2-subunit is a highly glycosylated extracellular protein that is attached to the membrane-spanning d-subunit by means of disulfide bonds. The a2-subunit provides structural support whilst the d-subunit modulates the voltage-dependent activation and steady-state inactivation of the channel Ca2+ Channel a-Subunits Molecular Composition : Ca2+ Channel a-Subunits Molecular Composition Ca2+ Channel a-Subunits Structural elements of function : Ca2+ Channel a-Subunits Structural elements of function Ca2+ Channel a-Subunits Genetic Disorders : Ca2+ 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 Skeletal Ca2+ Channel a-Subunits Genetic Disorders : Skeletal Ca2+ Channel a-Subunits Genetic Disorders Hyperkalemic periodic paralysis Malignant hyperthermia Ca2+ 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 Channels : 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+ Channels Responsible for repolarization of the action potential and refractoriness (consequences for contractility and arrhythmias) Slide 40: 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) Slide 41: Leak K+ Channels Plateau (IKP) K+ channels “Leak” 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 Slide 43: 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 Slide 44: 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 eag KCNQ SK slo Kv eag erg elk Kv1 Kv2 Kv3 Kv4 Kv5 Kv6 Kv8 Kv9 Kir1 Kir2 Kir3 Kir4 Kir5 Kir6 Kir7 KCNK1 KCNK9 KCNK2 KCNK10 KCNK3 KCNK12 KCNK4 KCNK13 KCNK5 KCNK15 KCNK6 KCNK16 KCNK7 KCNK17 Kir SUR KCR1 minK MiRPs Kvb KChAP KChIPs NCS1 Coetzee, 2001 Voltage-activated K+ Channels : 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 Ito : 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 : Delayed Rectifier Currents IKr and IKs Delayed Rectifier Current : Delayed Rectifier Current Matsuura et al, 1987 Control Ca-free + Cd Two Types of Delayed Rectifiers : Two Types of Delayed Rectifiers Sanguinetti & Jurkiewicz, 1991 E-4031 550 ms 100 pA Compounds Blocking Delayed Rectifiers : Compounds Blocking Delayed Rectifiers Rapidly activating (IKr) E-4031 Dofetilide Sematilide MK-499 La3+ Slowly activating (IKs) K+ sparing diuretics Indapamide Triamterene K+ Channel a-Subunits Molecular determinants of gating : K+ Channel a-Subunits Molecular determinants of gating N-type inactivation pore S4 segment Slide 53: Kvb Subunits Accelerate Inactivation of Kv Channels Slide 54: Kvb Subunits Increase Expression Levels of Kv Channels Slide 55: Enhanced Surface Expression Slide 56: Kvb Subunits as Molecular Chaperones Slide 57: 3-Dimensional Structure of Kvb2 Slide 58: Kvb Confers Hypoxia-Sensitivity to Kv4 Channels 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) Slide 60: Effects of Frequenin on Kv4.2 Currents Kv4.2 + H2O Kv4.2 + Frequenin 0 5 10 15 20 * 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+ 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 : 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 : Voltage-activated K+ Channels Genetic Disorders 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 : Factors Promoting EADs Autonomic - increased sympathetic tone - increased catecholamines - decreased parasympathetic Metabolic - hypoxia - acidosis Electrolytes - Cesium - Hypokalemia Factors Promoting EADs : Factors Promoting EADs Drugs - Sotalol - N-acetylprocainamide - Quinidine Heart rate - Bradycardia Slide 72: 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 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 : 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+ ChannelsStructure : Inward Rectifier K+ ChannelsStructure Two transmembrane domains Pore No voltage sensor Slide 75: 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 Slide 76: 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 eag KCNQ SK slo Kv eag erg elk Kv1 Kv2 Kv3 Kv4 Kv5 Kv6 Kv8 Kv9 Kir1 Kir2 Kir3 Kir4 Kir5 Kir6 Kir7 KCNK1 KCNK9 KCNK2 KCNK10 KCNK3 KCNK12 KCNK4 KCNK13 KCNK5 KCNK15 KCNK6 KCNK16 KCNK7 KCNK17 Kir SUR KCR1 minK MiRPs Kvb KChAP KChIPs NCS1 Coetzee, 2001 Inward Rectifier K+ ChannelsGenetic Disorders : Inward Rectifier K+ ChannelsGenetic Disorders Inward Rectifier K+ ChannelsPharmacology : 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 Slide 79: 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 eag KCNQ SK slo Kv eag erg elk Kv1 Kv2 Kv3 Kv4 Kv5 Kv6 Kv8 Kv9 Kir1 Kir2 Kir3 Kir4 Kir5 Kir6 Kir7 KCNK1 KCNK9 KCNK2 KCNK10 KCNK3 KCNK12 KCNK4 KCNK13 KCNK5 KCNK15 KCNK6 KCNK16 KCNK7 KCNK17 Kir SUR KCR1 minK MiRPs Kvb KChAP KChIPs NCS1 Coetzee, 2001 Slide 80: 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 Release : 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 : Inward Rectifier K+ ChannelsGenetic Disorders Slide 86: Glibenclamide Blocks KATP Channels 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 Next Thursday : Next Thursday

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