Calcium Channel Pharmacology Calcium Channel Pharmacology Edited by Stefan 1. McDonough Marine 8ioiogicailAboratory Woods Hole, Massachusetts Springer Science+Business Media, LLC Library of Congress Cataloging-in-Publication Data ISBN 978-1-4613-4860-3 ISBN 978-1-4419-9254-3 (eBook) DOI 10.1007/978-1-4419-9254-3 ©2004 Springer Science+Business Media New York Originally published by Kluwer Academic/Plenum Publishers in 2004 Softcover reprint of the hardcover 1s t edition 2004 AII rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, record ing, or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Permissions for books published in Europe: [email protected] Permissions for books published in the United States of America: perm;ss;ons@wkl,p.com PREFACE Voltage-gated calcium channels are critical regulators ofcytoplasmic levels of calcium, the universal signaling ion. As such, calcium channels trigger a wide range of cellular functions, from muscle contraction to neurotransmitter secretion, and are important players in human disease. Prominent in the nervous, cardiovascular, and endocrine systems, members of the calcium channel family are targets forexisting antihypertensive and anticonvulsant drugs. In addition, they are emerging targets for drugs to treat an extraordinarily diverse group of disorders, including pain, cerebral ischemia, cardiac arrhythmia, andmigraine. This book reviews the compounds that target individual calcium channel subtypes andthe cellular and behavioral functions governed by each different channel. Itcontains information for basic scientists using calcium channel antagonists as experimental tools, for behavioralists studying animal models of human disease, and for pharmaceutical scientistsinterested increating the nextgeneration ofcalcium channel-targeteddrugs. Several factors make an entire book on calcium channel pharmacology timely. Within the past several years, entire new families of calcium channels have been cloned, and genomic analysis finds ever more splice forms of the same gene with distinct physiology andpharmacology. Calcium channels havebeen found tobe thegeneticcause of several forms of human disease, and for almost all calcium channels, the behavioral phenotypes of mice carrying targeted gene disruptions have now been reported. Many more drugs and neurotoxins that target calcium channels are available than is generally known,andthebest-known calcium channelblockers are understood ingreatmechanistic detail, sufficient to serve as a guidepost for the development of future drugs. Calcium channels are increasingly tractable drug targetsjust as their contributions to a surprising diversityofdisorders andofcellular functions havebecome clear. Targeting drugs to individual calcium channels, and even to individual channel gatingstates,offers both achallengeand anopportunity, scientificallyandclinically.Itis ourhopethatinformation presented here willhelpclarify theissuesandthetools. I am deeply grateful to all my fellow authors and especially to the chapter lead authors:Drs.Steffen Hering,DavidTriggle, CharlesCohen, Raymond Norton, EricErtel, Erika Piedras-Renteria, Tsutomu Tanabe, Yasuo Mori, Gerald Zamponi, and Diane Lipscombe.Yourcontributionshave madethebook. StefanMcDonough v CONTENTS Introduction ix StefanI.McDonough ConceptsofState-DependentPharmacologyofCalciumChannels I EvgeniN.Timin,StanislavBerjukow,and Steffen Hering PharmacologyofCayl (L-Type) Channels 21 David1.Triggle The TherapeuticUtility ofTargetingCay2Channels 73 Charles J.Cohen and Richard L.Kraus PeptideToxin InhibitionofVoltage GatedCalciumChannels:Selectivity and Mechanisms 95 StefanI.McDonough CalciumChannelBlocking Polypeptides: Structure,Function,and Molecular Mimicry 143 Raymond S.Norton,JonathanB. Baell,andJames A. Angus PharmacologyofCa.,3(T-Type) Channels 183 Eric A.Ertel CellularFunctionsofCalciumChannelSubtypes 237 Erika S.Piedras-Renteria,Paul G.Mermelstein,and GeoffreyS.Pitt Genetic Approachestothe ElucidationofCalciumChannelFunctionsinVivo.....277 Hironao SaegusaandTsutomuTanabe CalciumChannelMutations and Associated Diseases 303 Yasuo Mori,Yuko Itsukaichi,Motohiro Nishida,andHiroaki Oka ModulationofHigh Voltage-ActivatedCalciumChannelsbyGProtein- CoupledReceptors 331 AaronM.Beedle andGerald W.Zamponi AlternativeSplicing inVoltage GatedCalciumChannels 369 DianeLipscombeand Andrew1.Castiglioni Index 411 vii INTRODUCTION Voltage-gated calcium channels are integral membrane proteins that open in response to electrical depolarization ofthe plasma membrane and mediate the entry ofcalcium ions into the cell. Expressed in every excitable cell, including neurons, myocytes, and pancreatic ~ cells, calcium channels form the most efficient molecular link between membrane depolarization and intracellular biochemical signaling (Catterall, 2000). At the cellular level, calcium influx mediated by voltage-gated calcium channels governs diverse physiological functions. Muscle contraction, neurotransmitter and hormone release, regulation of membrane excitability (either directly or via calcium-activated potassium and chloride channels), and the regulation ofgene expression all depend on calciumchannel function. Calcium channelsarecurrent orpotentialtargets fordrugs treating a wide variety of common disorders, including forms of hypertension, pain, epilepsy, migraine, cerebral ischemia, and cardiac arrhythmia, and calcium channel dysfunction underlies rarer diseases such as hereditary forms of ataxia and night blindness. The many functions that depend on calcium channels are not governed by the expression ofa single channel. Rather, calcium channels come in many biophysically, pharmacologically, and biochemically distinct subtypes. Recent evidence shows that different subtypes ofcalcium channels support distinct physiological functions, at levels from the single cell to the entire organism. This book deals with calcium channel pharmacology-the study of the physiological roles of different calcium channel subtypes and ofthe drugs that distinguish among them. The two goals are interrelated: selective drugs are indispensable for the discovery ofsubtype-specific functions,and the broad finding that calcium channel subtypes govern different functions drives the discovery and further refinement ofselective drugs. In addition, characterization ofthe molecular activity of clinically useful drugs, whether against single calcium channel subtypes, multiple calcium channel subtypes, or a broad spectrum of ion channels, contributes greatly to the understanding and treatment ofdisease. The purpose ofthis book is to review the selectivity and biomedical significance ofdrugs that distinguish amongdifferent calcium channel subtypes. Diversity among calcium channels was first noted in electrophysiological experiments. Multiple components ofcalcium channel currents within single cells were found to be separable on the basis of biophysical or pharmacological properties (Hagiwara et aI., 1975; Carbone and Lux, 1984a,b; Bean, 1985; Nowycky et aI., 1985) and were named with a descriptive letter (Nowycky et aI., 1985). In parallel with ix I INTRODUCTION Table1. Calciumchannela.subunitsandcorrespondingcalciumchannelcurrents. Family al subunit Formername Ca2+current Cav1.l alS Cav1.2 ale L-type Cavl.3 aID CaviA alF Cav2.1 P(orP/Q)-type alA Cav2.2 alB N-type Cav2.3 alE R-type(some) Cav3.1 alG Cav3.2 alH T-type Cav3.3 an Calcium channel a, subunits arelistedusingtheagreednumeric nomenclatureestablished by sequencehomology (Ertelet al., 2(00) together withthepreviousletternomenclature. See chapterbyLipscombe andCastiglioni fora,subunitgenetreeandforterminologyofsplice variantsofarsubunits. Notallresistant orR-type currents result fromCa,.2.3 channels;see chapterbyMcbonough fordetailed description ofR-type channels andforexplanation ofP typeandP/Q-typechannels. y NMffi c N c Figure I. Cartoon hydropathyanalysisofcalciumchannelalt ~.a14'5.and'Ysubunits. Theal subunitconsists of fourhomologous membrane-spanning domains (I-IV)eachencompassing a helical structures in domains termedSI through S6, with a pore-loop region re-entering the membranebetweenSSandS6. SeeJiangetal.,2003a,bforX-raystructureofavoltage-gatedK+ channel,atetramerhomologoustothefourinternaldomainsoftheal subunit,thatshowstheactual location,orientation,structure,andboundariesofthea-helicesandhowtheycombinetoformthe channelvoltagesensor. The~ subunitisintracellular;theal subunitisextracellularwithdisulfide linktothetransmembrane 0subunit,andthe'Y subunit is predictedtohavemultiple membrane spanningdomains. INTRODUCTION xi physiological studies, biochemical and molecular experiments revealed a diversityofcalciumchannelproteins and genes,starting with thepurification (Curtis and Catterall, 1984)andcloning (Tanabe etaI., 1987)oftheskeletal muscle dihydropyridine receptor. A full calcium channel protein is composed of a pore-forming.UI subunit, which consists of a fourfold internal repeat of the voltage-gated channel superfamily motif (Hille, 2001),plus additional ancillary ~, U20, and y subunits (Figure 1). The U2 and 0 subunits are derived from the same gene, with the product split into two peptides linked by a disulfide bond (de Jongh et aI., 1990). Biochemical and physiological evidencesuggests that mostcalciumchannels are composed ofu" ~, U20,andpossiblyy subunits in 1:1:1:1stoichiometry (Takahashi et aI., 1987;McEnery et aI., 1991;Witcher et aI., 1993;Liu et aI., 1996;Kang et al., 2001). Involvement and function ofthe y subunitisanareaofactiveresearch (MossetaI.,2002). The multisubunitcompositionof T-typechannelprotein islikewisecontroversial (seeErtel,thisvolume). As ofthiswriting,ten UI geneshave been discovered and are named numerically according to sequence similarity (Ertel et aI., 2000). These ten genes may represent the complete suite ofUI subunits, with the possible exception ofa cryptic cardiac calcium channel found after knockout ofthe Cay1.2uI gene (Seisenberger et aI., 2000). Four ~ subunits (Castellano and Perez-Reyes, 1994),four U20 subunits (Klugbauer et aI., 1999; Qinet aI.,2002),and eightysubunits (Klugbauer et aI.,2000;Chuet aI.,2001;Moss et aI., 2002) are currently known. T-type channels excepted, it seems likely that any ancillary'subunitcan forma channel withany UI subunit (Walker and De Waard, 1998), although not all combinations have been tested. In addition to modifying physiological properties of the UI subunits, ancillary subunits likely have additional functions in trafficking!surfaceexpression ofcalciumchannels (Dolphin etaI.,1999;Brodbecketal., 2002) and, surprisingly, of non-calcium channel proteins in at least some cases (HashimotoetaI., 1999;ChenetaI.,2000). Terminology of different calcium channel subtypes is complex. To a first approximation,themainphysiological andpharmacological properties ofagivenchannel are determined by the molecular identity ofthe UI subunit, most ofwhich have been linkedconvincingly withcorresponding channelsubtypes innativetissue(Table 1). Cay3 T-type channels are mostly distinguished by their biophysical properties (see Ertel, this volume), but for Caylor Cay2 channels pharmacological profiles are often used as a guide to the UI subunit. Channels sensitive to dihydropyridines are generally called L type, and those inhibited with high affinity by the peptide conotoxin ro-CTx-GVIAare called N-type. Other subtypes include P-type (or P/Q-type) channels, some forms of which are highly sensitive to the peptide toxin ro-Aga-IVA from spider venom. R-type channels were first referred to as those channels resistant to combined inhibition by blockers ofother channels and likely represent a heterogeneous group ofchannels. For clarity, use of "type" names for channels should be qualified with either molecular characterization of the gene involved or with the pharmacological procedure used to isolatethecurrent. Thisbook isgroupedbroadly intotwoparts;individual chapters arecomplementary, but can stand individually. The first part covers the specificity and selectivity ofdrugs that target various classes ofcalcium channels. Like all ion channels, calcium channels are moving targets. That is, the channel protein itself physically changes shape as depolarizationevokestransitsamongclosed,open,andinactivated states,and,moreoften xii INTRODUCTION thannot,thepotencyofa givendrugdepends dmmaticallyontheprecisegatingstateof thechannel. Thisstate-dependentaffinitycancomplicateuseofdrugsasscientifictools, buttheabilitytotargetspecificchannelgatingstatesoftencontributesgreatlytoadrug's therapeuticprofile. Inthefirst chapter,Timin, Berjukow,andHeringdiscussmodelsfor state-dependent drug affinitythat underlieall discussion of pharmacology, including open-channelblock,themodulated receptormodel,anddrug-inactivationsynergism. In thesecondchapter,TrigglereviewsthepharmacologyofCa.IL-typechannels,thebest developed pharmacology. Thusfar, thesuccessof smallmolecule blockersfor L-type channels has not been repeated for the Cav2 family of P-type, N-type, and R-type channels. In thethird chapter, Cohenand Krausreviewthe pharmaceutical interestin findingsuchmolecules to treat pain and migraine and as neuroproteetive agents. The mostspecificblockersforchannelsoftheCav2familyarepeptidetoxins,mostlyderived fromthevenomof poisonous animals. Peptidetoxininhibition ofcalciumchannelsis dealtwithintwochapters,focused respectivelyonchannelsandontoxins. In thefourth chapter, McDonough reviews toxin selectivity, mechanisms of action, and channel binding sites; in the fifth chapter, Norton, Baell,and Angusreviewthe structureand "structure-functionrelationsofpeptidetoxins,theuseofsometoxinsthemselvesasdrugs to treatpain, andthedevelopment ofsubtype-selective smallmolecules basedon toxin structure. In the sixth chapter, Ertel reviews the physiology, pharmacology, and modulationofT-typecalciumchannels,thenewestfamily tobecloned(perez-Reyes et al.,1998). The second part of the book covers, broadly, the physiological functions of individual calcium channel subtypes. In the seventh chapter, Piedras-Renteria, Mermelstein,andPittdiscussthecouplingofcalciumchannelstofundamentalprocesses withinthe cell, especially withinneurons: neurotransmitter release, neuronalplasticity, geneexpression,theactivationofcalcium-activatedpotassiumchannels, andinteractions withcalmodulinand CaMkinase II. Intheeighthchapter, SaegusaandTanabediscuss thephenotypes ofcalciumchannelknockout mice. Suchmiceoftenhaveastonishingly specificphenotypes and provide insights intothe distinct roles or overlapof calcium channelsubtypesatthebehavioral level. Knockout micearealsovaluableasmodelsof humandisordersanddisease. In theninthchapter, Morl, Itsukaichi, Nishida, andOka review human and animal diseases resulting from mutations in calcium channels, includingmigraine,ataxia,andformsofepilepsy,withaviewtowardsexplaininggeneral mechanisms of how molecular defects cause disease. Although channelopathies generally representrare forms of humandisease(Ashcroft, 2(00), there is hope that understanding the geneticforms of diseasewill lead to treatments for more common forms. Many existingclinicaldrugsthatexerteffectsoncalciumchannelsdosoindirectly, via G protein-coupled receptors. In the tenth chapter, Beedle and Zamponi review mechanisms of G protein inhibition of high voltage-activated (non-T-type) calcium channelsand summarize knowledge aboutthe neurotransmitter and hormonereceptors thatcoupletocalciumchannelsindifferentcells. Finally,perhapsthenewestfrontierin calcium channel pharmacology is alternative splicing. In the eleventh chapter, Lipscombe and Castiglioni present the genomic structure of calcium channels, the mechanisms by which potentially hundreds of splice variants are produced, and the contributionsofalternativesplicingtocalciumchannelphysiologyandpharmacology. It is of great importance to noterecentwork fromthe MacKinnon laboratory, the high-resolution X-ray structure of a bacterial voltage-activated K+ channel from INTRODUCTION xiii Aeropyrumpernix (Jiang et al., 2003a; Jiang et al., 2003b; Ruta et al., 2003), work that appeared as this book was in proof The structure of the K+ channel, a tetrameric (MacKinnon, 1991)assembly homologous to the fourfold internal repeat ofthe calcium channel, has major implications for calcium channel topology and pharmacology. By analogy, the predicted transmembrane helices (Figure 1)are not all transmembrane, not allborderedas marked,and notpositionedasshown,butratherareburied withinthe membrane at a variety ofangles. Opening ofthe K+channel in response to voltage is accomplished by movement from internal- to external-facing sides of the plasma membrane of a charged helix-turn-helix paddle, made up of the latter part of the previously-named S3 helix plus S4. Paddle movement is transferred to opening ofthe channel via other helices. For the many drugs and toxins that are voltage-dependent inhibitors ofcalcium channels, drugaccess to thechannel, the mechanisms ofinhibition, and channel mutations affecting drug affinity should be viewed in light ofthis gating mechanism,and modelsofchannel-drug interactionscannowbe viewed morephysically ratherthantheoretically. ACKNOWLEDGMENTS IthankDr.DianeLipscombeforhelpfulcomments. Stefan1.McDonough WoodsHole,Massachusetts REFERENCES Ashcroft,F.M.,2000,JonChannelsandDisease,AcademicPress,SanDiego,London. Bean,B.P.,1985,Twokindsofcalciumchannels incanineatrialcells.Differences inkinetics,selectivity,and pharmacology,J GenPhysiol86:1-30. Brodbeck,J.,Davies,A.,Courtney,J.M.,Meir,A.,Balaguero,N.,Canti,C.,Moss,F.1.,Page,K.M.,Pratt,W. 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