Table Of ContentCalcium 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
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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
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