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ReceWor sites for Ca*+’ channel antagonists William A. Catterall and J&g Striessnig C$+ channel antagonist drugs inhibit voltage-gafed Ca’+ channels in many different cell fypes. Inhibition of Ca 2+ channels in smooth muscle and cardiac muscle cells by these drugs is valuable in the fheTUQy of a wide range of cardiovascular disorders including hypertension, atria1 arrhythmia and angina pecforis. Additional uses under evahiafion are protection against ischemic damage during myocardial infarction and stroke and in a wide range of other conditions. Further understanding of the sites and mechanisms of action of Cd+ channel antagonists, as described in this review by Bill Catterall and &rg Striessnig, will provide new insig?lf info the design of novel therapeutic agents acting on Ca’+ channels and provide further understanding of Ca2+ &annel stnccture and function. Caz+ channels have been typed according to their physiological properties’. Low-voltage-activated, or T-type, Ca” channels are activated at relatively negative membrane potentials, have a small single-channel conductance, and mediate a transient Ca” current that is important in determining the frequency of action potential generation in neurons and cardiac muscle cells. High-voltage-activated Ca2+ channels require a more positive membrane potential for activation and include L, N and P channel types. N- and P-type Ca2’ channels are inactivated at membrane potentials more positive than 40 mV and have an intermediate single-channel conductance compared with T and L types. They are expressed primarily in neurons where they play an important role in neurotransmitter release. L-type Ca2+ channels are not strongly inactivated by depolarization to -40 mV, have the largest single-channel conductance among the voltage-gated Ca2’ channels, and have both slow voltage-dependent and more prominent Ca2+-dependent mechanisms of inactivation. L-type Ca2+ channels are the primary type in muscle cells and are responsible for the inward movement of Ca2+ that initiates contraction in cardiac and smooth muscle cells. They are the molecular target for

the actions of the therapeutically useful Ca2’ channel antagonist drugs. Pharmacology of L-type channels The functional properties of Ltype Ca2’ channels are modulated in a complex manner by Ca2’ channel antagonist drugs that act at multiple receptor sites. Members of each of the three main chemical groups - the phenylalkylamines, benzothiazepines and dihydropyridines - are widely used in the therapy of cardiovascular disorders. The phenylalkylamines are a structurally diverse group of compounds exemplified by verapamil (Fig. 1). Inhibition of Ca*+ channels by verapamil and other

phenylalkylamines

phenylalkylamines is greatly enhanced by repetitive stimulation with depolarizing voltage pulses that activate the channels2r3. These results indicate lhat the drugs gain access to their receptor sites more rapidly when the channel is in the open state. Quaternary derivatives of phenylalkylamines are effective inhibitors of Ca’+ channels only when applied to the intracellular surface of the channel* (Fig. 2). Evidently, the receptor site for phenylalkylamines is exposed only on the intracellular side of the channel protein. The requirements for channel activation and intracellular application of impermeant drugs suggest that the phenylalkylamines may enter the transmembrane pore of the Ca2+ channel from the intracellular side, bind to their receptor site, and occlude the pore. In contrast to the phenylalkylamines, the dihydropyridines are of the primarily modulators voltage-dependent gating of Ca2’ chax’mels. Dihydropyridines can act either as channel activators or as inhibitors; enantiomeric pairs often have opposite influences on chbnnel function5. Binding of the dihydropyridine Ca2+ channel antagonists is not strongly influenced by repetitive activation of the Ca*+ channe123, indicating that they do not bind preferentially to the open state of the channel. However, the binding and

dihydropyridines

verapamil nitrendipine HCI

ludopamil

W, CH H&’

benzothiazepines isradipine

W. A. Catterall is Professor and Chairman, and I. Striessnig wus Visiting Scientist, at the

Department of Pharmacology, SI-30, University of Wushington, Seattle, WA 98195, USA. I. Striessnig is currently at the Institut ftir Biochemische Pharmakologie, Uniuersitiit Innsbruck, A6020 Innsbruck, Austria. @

1992. Elsevier Science Publishers Ltd (UK)

dH3

diltiazem Fig.1.Representafive structuresof fhe three c/asses of Ca2+ channel antagonist drugs.

TiPS -June

2992 lVol.131

257

phenylalkylamine

100

1

!

“1P

0 E t a

.S 3

dihydropyridine

ouside

•-o-m*.~++.Co-.

\

inside O\O,

50-

0+

\ inside

“\

84

O\ O\O

0 0

I 60

Jl120 480

150 0

Time(s)

I 20

I 40

I 60

Time (s)

Fig. 2. Intracellular and extracellular actions of permanently charged phenylallrykmines and dihydropyridines. The permanent/y charged quaternary phenylalkylamineI3890 was applied intracellulariy at I PMor extracellu~ady at 100 lrrr~to single guinea-pig ventricular myocyfes at time 0 and againat thearrow,and the effect on the plateau phase of the cardiac action potential was measured. Redrawn from Ref. 4. The permanently charged quaternary dih-gdropyridineSD2207180 was applied intracellular/y or extrace//u/ar/y at 1 w to single guinea-pig venhicular myocytes at time 0 and Ca + currents were recorded by the whole-cell voltage-clamp method.Redrawn fromRef.9.

inhibition of the Ca2+ channel by dihydropyridines is enhanced by prolonged depolarization6. These results indicate that the dihydropyridine Ca2+ channel antagonists bind with high affinity to inactivated Ca2+ channels6. Dihydropyridines modulate the function of L-type Ca2+ channels by favoring distinct modes of channel gating. Activators favor gating mode 2 with long singlechannel openings whereas inhibitors favor gating mode 0 in which the channel fails to open in response to depolarization’. Dihydropyridines bind to their receptor site from the extracellular surface of the Ca2+ channel. Protonated tertiary dihydropyridines act more rapidly from the extracellular side of the membrane and can be deprotonated by external hydroxyl ion, causing rapid dissociations. Quaternary derivatives of dihydropyridines are inactive when perfused inside the cell but rapidly block Ca2+ currents from outside’ (Fig. 2). Dihydropyridines are strongly bound by lipid bilayers and appear to be concentrated along the surface of the bilayerlo. Evidently, the dihydropyridine receptor site is located in a hydrophobic region of the Ca2+ channel near the outer surface of the membrane. Molecular properties of L-type channels Ca2+ L-type voltage-gated channels are present in high con-

centration in the transverse tubules of vertebrate skeletal muscle. Their high density has enabled purification of these Ca2’ channels using their high-affinity binding of dihydropyridine Ca2+ channel antagonists as a specific label, and they have served as a model for structure-function studies of L-type Ca2+ channels. These channels are a complex of five protein subunits (Fig. 3) (reviewed in Ref. 11). The cul subunit is the central functional component of the complex. Its mRNA encodes a protein of 212 kDa having four homologous domains with six proposed a-helical transmembrane segments in each, like Na+ channel LX subunits’2 (Fig. 3). Distinct genes encode cul subunits that are expressed in heart, smooth muscle and brain’. The oil subunit can function as a voltage-gated ion channel when expressed in appropriate recipient cells13,“. arl Subunits are present in a complex with an intracellularly disposed p subunit of 55 kDa, a glycosylated transmembrane y subunit of 30 kDa, and a disulfide-linked glycoprotein complex of or2 and 6 subunits of 143 and 27 kDa, respectively. Phenylalkylamine receptor site The phenylalkylamine receptor site on purified skeletal muscle Ca*’ channels can be covalently labeled with ludopamil, a photoreactive arylazide phenylalkylamine15,16 (Fig. 1). Ludopamil binds to its receptor site on puri-

fied skeletal muscle Ca*+ channels with high affinity before photoactivation and is specifically incorporated into the al subunit of the Ca” channel after photoactivation. The site of photoincorporation has been determined by mapping of labeled peptide fragments of the arl subunit with antipeptide antibodiesI’ (Fig. 4). Specifically labeled or1 subunits isolated by SDS-PAGE were cleaved at lysine and arginine residues with trypsin to yield a single photolabeled peptide of 9.5 kDa. This labeled peptide fragment was identified by immunoprecipitation with a panel of sitedirected antipeptide antibodies (Fig. 4). Only antibodies against the peptides CP133s13~ and immunoprecipitated cp1382-1400 the labeled peptide, placing the site of covalent labeling of the crl subunit within a 9.5 kDa peptide residues containing fragment 1339-1400. Further cleavage at glutamate and aspartate residues with staphylococcal V8 protease yielded a 5 kDa fragment that was recognized by antibodies against CP1382-1400 (Fig. 4) butC;t antiagainst bodies 1339-1354 Considering the size of these peptide fragments and the location of possible cleavage sites for trypsin and V8 protease, the site of covalent incorporation into the al subunit can be localized to the 42residue segment extending from Glu1349 to Trp1391. This region includes transmembrane segment S6 in domain IV of the or1subunit

TiPS -June 1992 [Vol. 131

Ca*+

Fig. 3. Stnrcturalmode! of L-type Ca 2+ channel from skeletal muscle. Lett: subunit structure of L-type Ca” channel from skeletal muscle transverse tubules as determined from biochemical studies. P, phosphorylation site. Below: proposed folding patterns of the polypeptide components of the ske/eta/ muscle Ca2+ channel as predicted from the amino acid sequences deduced from CDNA cloning and sequencing. Recognition sites of three antipeptide antibodies direc!ed against amino acid sequences beginning at residues 1025, 7339 and 1382 are indicated (boxes). See Ret. 11 for ref. details.

outside

5.9 4.8 637

743

1092

1339

1392

1505

Anti-peptide antibody trypsin digest

and several adjacent intracellular and extracellular amino acid residues (Fig. 3). Because quatemary phenylalkylamines reach their receptor site from the intracellular surface of the membrane*, it is likely that the intracellular end of segment

VE protease digest

IVS6 and the adjacent intracellular amino acid residues contribute to formation of the phenylalkylamine receptor site. Since inhibition of Ca*+ channels by phenylalkylamines is greatly accelerated by depolarizations that open the channels2*3, the

Fig. 4. Identification of peptides covalently labeled by the phenylafkylamine ludopamil. Left: purified skeletal muscle Ca2+ channels, photoaffinity labeled with [%Jludopamil, on SDS-PAGE. Left lane, control, reveals specific labeling of the 175 kDa cul subunit. Right lane, photolabeled rul subunit digested with trypsin, reveals a sing/e covalent/y labeledpeptide of 9.5 kDa. Middle: immunoprecipitation of the 9.5 kDa labeled peptide with several anti-peptide antibodies directed against amino acid sequences beginning at the indicated residues in the cul subunit. Percent immunoprecipitation versus undigested ~1 subunit is shown. Right: fhe 9.5 kDa ttypsin fragment further digested with staphylococcal V8 protease resulted in a pair of 4.8 and 5.9 kDa peptides (left lane). Both VB peptides were immunoprecipitated by anti-CP,3e2-,400 (right lane) Experimental details in Ref. 17.

phenylalkylamine receptor site may reside within the intracellular opening of the transmembrane pore and bind phenylalkylamines rapidly and with high affinity only when the pore is open. The amino acid residues near the intracellular end of segment IVS6

TiPS -June 1992 [Vol. 23)

259

Fig. 5. Pore-blocking model forphenylatkytamine binding and action on Cazf channels. Left: predicted transmembrane folding of the membrane-associated core of the K+ channel. Closed circle illustratesapproximate position of extracellular amino acid residues required for charybdotoxin binding and block of K+ channels. Open circles indicate approximate position of amino acid residues required for block of K’ channels by intracehlarand extracellufar tetraethylammomum ion. Redrawn from Ref. 18. Right: sketch of a cross-section through the Ca” channel cl subunit illustrating formation of central transmembrane pore lined by peptide segments between transmembrane u-he/ices S5 and St? of domains I and IV as proposed for K+ channels. The peptide segment covalent/y labeled by verapamil derivatives is illustrated by the open box. VE R indicates possible position of binding of verapamil as an intracellular pore bfocker.

may therefore also contribute to formation of the intracellular mouth of the transmembrane pore of the Ca2+ channel. This segment is highly conserved among different Ca2+ channels’. Pore-blocking model for phenylalkylamine action Recent studies of voltage-gated KC channels have identified amino acid residues in the region between o-helical transmembrane segments S5 and S6 that are required for binding of both extracellular and intracellular pore blockers and for normal ion selectivity (reviewed in Ref. 18). As illustrated in Fig. 5, these results have led to the proposal that this 18-residue peptide segment folds across the membrane and lines the pore of the K+ channel in an extended nonhelical conformation such as a twostranded antiparallel P-sheet’s,19. Amino acid residues required for extracellular block by the peptide blocker charybdotoxin pore and by tetraethylammonium ion, and residues required for intracellular block by tetraethylammonium ion, were identified by site-directed mutagenesis and functional expression. Amino acid residues throughout this region are required for normal ion

selectivity. These results support the designation of this region as the pore-lining segment of the K+ channel. The similarity of structure among Na+, Ca2+ and K+ channels implies that the corresponding segments of all three channel types line the walls of their transmembrane pores. If this segment does indeed line the transmembrane pore, the adjacent transmembrane ar-helices 55 and S6 are likely to surround the pore lining. This arrangement would place the ends of the %I transmembrane segments around the intracellular and extracellular openings of the transmembrane pore. Binding of phenylalkylamines to a receptor site formed in part by sequences near the intracellular end of transmembrane segment IV!% could serve to block ion movement into and out of the transmembrane pore (Fig. 5). Therefore, localization of the phenylalkylamine receptor site in this position supports the proposal that these drugs act as intracellular pore blockers which directly interfere with ion movement. Dihydropyridine receptor site The dihydropyridine receptor site on skeletal muscle Ca2+ channels can be covalently labeled

with multiple photoreactive reagents. The arylazide azidopine2“ and the diazirine diazipinezi have nitrene- or Carbene-generating photoreactive groups on a sidechain that can extend up to 14 A from the dihydropyridine ring (Fig. 1). The dihydropyridines isradipine, nitrendipine and nifedipine (Fig. 1) are intrinsically photoreactive and can be photoactivated to label covalently their receptor site without derivatization. It would be expected that isradipine, nitrendipine and nifedipine would label the core of the dihydropyridine receptor site because their photoreactive moiety is intrinsic to the binding center of the drug molecule while azidopine and diazipine might label more peripheral regions of the binding site. The initial studies of covalent labeling of Ca2’ channels in purified skeletal muscle transverse tubules by dihydropyridines resulted in specific photoaffinity labeling of proteins of 155-175 kDa with multiple dihydropyridine photolabels20z. Subsequent work showed that this labeled protein was the 01 subunit of the skeletal muscle Ca2+ channe1’6,~26 The site of covalent labehng of the 01 subunit has been studied with two different experimental strategies. Regulla et ~1.~’ photolabeled large quantities of pu& fied skeletal muscle channels, isolated the labeled 01 subunits, cleaved them by exhaustive digestion with proteolytic enzymes, purified the labeled reversed-phase peptides by HPLC, and determined the amino acid sequence of the major peptides in each labeled fraction by microsequencing procedures. The major peptides sequenced were derived from the hydrophilic intracellular C-terminal region of the 01 subunits including residues 1390-1399, 1410-1420 and 1428-1437. Amino acid sequences in the intracellular N-terminal region were also detected. Based on these results, it has been proposed that the dihydropyridines may bind to a site in the intracellular C-terminal region of the 01 subunit of the Ca2’ channel’“. In view of the physiological results showing that dihydropyridines must approach their receptor site from the extracellular surface’,‘, it seems unlikely that

TiPS -June

260 these peptide segments from the intradhhr N- and C-terminal domains can contribute directly to the receptor site mediating the inhibition of Ca*’ channels by the dihydropyridines. Moreover, in light of the evidence indicating that the dihydropyridine receptor site is in a hydrophobic environmen@*, it would be surprising if it were composed of predominantly hydrophilic peptides. Thus, it appears that these traditional protein-sequencing methods are not able to identify the components of the dihydropyridine receptor site. If this site is strongly hydrophobic as expected, it is likely that its component peptides cannot be easily recovered from hydrophobic reversed-phase HPLC columns by standard elution procedures. Specificahy labeled peptides from the core of the dihydropyridine binding site may not be recovered using these methods. An alternative approach to identification of the site of covalent attachment of photoreactive ~y~p~~nes to their receptor site is mapping the labeled peptides with sitedirected antipeptide antibodies. This method avoids the problem of 10s~ of hydrophobic peptides by maintaining samples continuously in the presence of appro-

F@ 6. ~~~~ of pepttdes labeled by the ~~~~ azidq&e and fsradipine. Pudfied sketatai moscfe Caz’ thanfl& were photokibekxl with PHjazidopine (a,~) or rHlisradiptne (b,d): the Meted al subunit wa& isolated bi WSDS-PAGE and treated with endopmteinase Lys-C @,b) a,

priate detergents. The antibody mapping procedure has been applied to al subunits specifically labeled with diazipine2’, azidopine and isradipine”. Cleavage of ~1 subunits covalently labeled with all three reagents results in the production of two separate peptide fragments from the ~~smernb~e regions of domains III and IV. For example, cleavage of azidopineand isradipine-labeled arl subunits at lysine residues with ~dop~teinase lys-C yields a peptide of 10.6 kDa recognized by antibodies against ~~1025-1040 and one of 7.6 kDa recognized by ar~ti-CPrs3+~=, as illustrated in Fig. 6. These results show that amino acid sequences in or near transmembrane segment S6 of domain III that are recognized by anti-CPi0z5-~~ and in or near S6 of domain IV that are recognized by anti-CP,s,,,,,, both participate in formation of the dihydropyridine receptor site. Further cleavage of the photolabeled peptides from domain III at both lysine and arginine residues with trypsin reveais differential labeling of adjacent peptide segments in domain III by azidopine and diazipine in comparison to isradipine. DiazipineZo and azidopine (Fig. 6), which have photoreactive groups at a distance

10.6

P0

a

2992 [Vol. 131

from the binding center, label 3.0 and 4.4 kDa peptides corresponding to amino acid residues 989-1022 of the extracellular loop in domain III. In contrast, isradipine, whose photoreactive moiety is intrinsic to the binding center of the molecule, covalently labels a 7.3 kDa peptide corresponding to residues 1023-1088 that includes transmembrane segment IIIS6 and adjacent intracellular and extracellular residues. Both these labeled fragments are contained within the 10.6 kDa fragment produced by trypsin cleavage (Fig. 6). The antibody mapping experiments give a different view of the localization of the dihydropyridine receptor site that is more consistent with previous pharmacological and physiological results (Fig. 7). The photolabeling results suggest that the core of the dihyd~p~~ne receptor site is formed in part by the extracellular end of transmembrane segment IIISG, which is preferentially labeled by isradipine. Transmembrane segment IVS6, which is a secondary site of photolabeling for all three drugs, may be located near segment IIIS6 in the folded structure of the arl subunit and contribute to formation of the dihydropyridine receptor site. Drugs like azidopine and

300

250

.10.6

b

~~~~~~~~(C~) to cleave at &sine and arginine rasidu*

separated by SDS-PAGEand quantified by slicJr?gthe get and counting radioactivity in 15 20 25 30 each slice. Labelad psptides were immunoprecipitated with anti-peotide antibodies. Recogni&n of the smaiiek iabeled peptide d fmgments by tha ~.-~p~ etch iden* their position iir- the amino a& SequenCe of the cut subunit. a: antiCP,-7m (O-0) twxxtnizes a 10.6kDa Lya-C fragm&t I&led-by azidopine that contains transmembrane segment f&6. (m-e) re&gnfzas a Anti-W,,354 7.6 kQa Lys-C fwment k&ted by azklopine that contains transmembrane segmant IVS6. b: isradipine photolabels the 10 15 20 25 30 same two peptide fragmentsas azidopine in Slicenumber Siicenumber a. c: . aner more complete cleavage wiIh tryps. ani%CP~~~~ recognizesa smeller7.3 kCa fragment tabeied by azidopine that contains t~s~rn~a~ segment MB Anti~KJW-~~ (A-A) ales two fragments. 4.4 and 3.0 ki& Iabefed by azidopine that are located on the e~~et~~r side of trrmamembranesegment I/W. d: after more complete cleavage wiih trypsin, anti-CP,025-,090n?cv$nias the sam6 7.3 kDa fragment /abeledby ismdfpinethat wasatso labeled by azidopine in pane/c, containing transmembranesegment 11156. By contrast, isradipine does not photolabel the hwosmall fragments recognized by anti-CP1011 _lo~ that are located on the extraoe/Mar side of lllS6.

TiPS -June 1992 [Vol. 231

261

n

diazipine which have long hydrophobic sidechains also interact with an adjacent subsite including amino acid sequences on the extracellular side of transmembrane segment lllS6. Thus, the extracelhrlar ends of transmembrane segments in two separate domains contribute to formation of the dihydropyridine receptor site. Allosteric domain-interface model for dihydropyridine action The dihydropyridines can be viewed as allosteric modulators of L-type Ca2’ channels since they modify gating functions which probably take place at some distance from their receptor site, and they bind in a state-dependent manner. Two important generalizations have arisen from studies of the three-dimensional structures of allosteric enzymes: allosteric control is usually exerted by changes in quatemary structure resulting from altered interactions at subunit interfaces, and binding sites for allosteric substrates and modulators are usually located at interfaces between subunits29-31. The allosteric modulation of phosphorylase by AMP and related nucleotides provides a particularly cogent model for the dihydropyridines because different ligands at this site can act either as allosteric activators or allosteric inhibitors of the enzyme. The AMP allosteric site is located at the interface between subunits, and ligand binding modulates the interactions between adjacent subunits resulting in allosteric control of the access of substrates to the active site32. In light of these studies of allosteric enzymes, it is provocative that photoaffinity labeling studies identify the extracellular ends of transmembrane peptide segments in domains Ill and IV (Fig. 7) as components of the dihydropyridine receptor site2’p2*. The four homologous domains of the Ca2+ channel arl subunit are likely to serve as symmetrical, covalently tethered structural units that are analogous to the separate subunits found in allosteric enzymes. By analogy with the regulatory sites of allosteric enzymes, the dihydropyridine receptor site may be located at the interface between domains Ill and IV and may affect

Fig. 7. A channels. as shown isradtpine

domain-interface

model for dthydropyrtdtne binding and action on Ca2+

Top: transmembrane folding modelof part of the Gas+ channel crl subunit

in Fig. S. The core of the dihydmpyrtdine-binding site spectticalty labeled by in the extracellular end of transmembtane helix MIS6 is indicated by the shaded ractangle, the secondary site of dihydropyeidine photolabeling in the extraceltular end of transmembrane helix IVS6 is indicated by the doRed rectangk, and the subsite photolabeled only by azidopine and diazipine on the extracellular side of transmembrane segment 11156 is indicateo’ by the hatchad curved bar. Bottom: proposed binding cleft for dihydropyddines footed at the extraceltular end of the interface between domains Ill and IV is illustrated in vertical (left, black bar) and horizontal (right, shaded) cross-sectional views of the ~1 subunit.

domain-domain interactions that are important determinants of activation gating (Fig. 7). The dihydropyridine antagonists inhibit Caz+ channel activity by inducing gating mode 0, in which activation in response to depolarizing stimuli is prevented. For K+ channels, two distinct forms of inactivation gating have been analysed at the molecular level. In N-type inactivation, the N-terminal region of the K+ channel is thought to bind to the intracellular mouth of the pore of the K+ channel shortly after opening and occlude it, causing

rapid inactivationaa. In C-type inactivation, a slower molecular transition leads to an inactivated state from which the channel cannot be activated by depolarization. The mechanism of this state unknown, but transition is hydrophobic amino acid residues near the extracellular end of transsegment S6 have membrane dramatic effects on this gating proce&. C-type inactivation of K+ channels is analogous to inhibition of L-type Car+ channels by dihydropyridine Ca2+ channel antagonists. In both processes, the channel is switched to gating

TiPS-june

262 mode 0 in which depolarization fails to cause activation. The comparison of results localizing Ctype inactivation of Ii’ channels and dihydropyridine binding regions of Ca’+ channels to analogous regions of the ion channel strwme suggest that dihydropyridines bind to the hydraphobic amino acids at the extracellular ends of transmembrane segments IRS6 and/or IVS6 and induce functional changes similar to C-type inactivation of K+ channels. Benzothiazepine receptor site The benzothiazepine receptor site has not been as completely analysed as the phenylalkylamine and dihydropyridine receptor sites. Recent photoaffinity labeling studies demonstrate that this receptor site, like those for the phenylalkylamines and dihydropyridines, is located on the crl subunip*36. Both azidobutyryl diltiazem and azido diltiazem specifically label only the ail subunit of purified skeletal muscle Ca2+ channels. These specifically photolabeled preparations of purified Cf’ channels are excellent substrates for analysis of the location of the benzothiazepine receptor site by the methods that have been established for the phenylalkylamine and dihydropyridine receptor sites. 0

cl

0

The receptor sites for dihydropyridines and phenylalkylamines have been located to two highly conserved regions of the Ca2’ channel ol subunit at which drugs can modify Ca2+ channel function in a highly selective manner. Comparison of the amino acid sequences of different Ca2+ channel ol subunits in the regions that are covalently labeled by phenylalkylamines and dihydropyridines indicates that these are highly conserved among L-type Ca*+ channels from skeletal muscle, heart and neurons, and also are We]] conserved in other neuronal Cazf channel types. The conservation of these regions in all known Ca” channels suggests that these may be pharmacologically privileged sites, that is, sites that are accessible to synthetic ligands, present in most Ca2+

susceptible to and channels, subtle modulation by drug binding. The dihydropyridines and phenylalkylamines in current therapeutic and experimental use were carefully selected for specificity for L-type Ca2+ channels in cardiac and smooth muscle. They are far less active on other types of Ca’+ channels. Nevertheless, the high conservation of their binding regions among known Ca” channels suggests that minor modifications of these basic drug structures may yield similarly selective, efficacious antagonists of other Ca2’ channel subtypes. Such agents acting on neuronal Ca” channels might be valuable in treatment of epilepsy, stroke and neurological diseases resulting from excitotoxicity. References 1 Tsien,R. W., Ellinor, P. T. and Home, 2 3 4 5 6 7 8 9 10 11 12 13 I4 15

W. A. (1991) Trends P~ranancoL Sci. 12, 34%354 Hondeghem, L. M. and Katzung, 8. G. (1984) Annu. Rev. Pknmracol. Toxicol. 24, 387-423 Lee, K. S. and Tsien, R. W. (1953) Nntttre 302.790-794 Hescheler, J., Pelzer, D., Trube, G. and Trautwein, W. (1982) PflrcgersArch. 393, 287-291 Triggle, D. J. and Janis, R. A. (1987) Atuuc. Rev. PltnrmncoL Toxicol. 27, 347-369 Bean, 8. P. (1984) Proc. Nat/ Acnd. Sci. USA 8l6388-6392 Hess, P., Lansman, J. B. and Tsien, R. W. (1984) Nature 311,538544 Kass, R. S. and Arena, J. P. (1989) J. Gen. Phvsiol. 93, 1109-1127 Kass, R., Arena, J. P. and Chin, S. (1991) J. Gcn. Physio!. 98, 63-75 Herbette,?L. G., Vant Erve, Y. M. H. and Rhodes, D. G. (1989) J. Mol. Cell. Cnrdiol. 21, 187-201 Catterall, W. A. (1991) Science 253, 1499-1500 Tanabe, T. ef RI. (1987) Nature 328, 3X3-318 Perez-Reyes. E. et RI. (1989) Nnfure 340, 233-236 Mikami, A. et al. (1989) Nnhrre 340, 230-233 Striessnig, J. ef al. (1987) FEBS Left. 212, 247-253

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16 Sieber, M., Nastainczyk, W., Zubor, V., Wemet, W. and Hofmann, F. (1987) Eur. I. Biockem. 167. 117-122 17 Striessnig, J.,. Glossmann, H. and Catterall, W. A. (1990) Proc. Nat/ Acad. Sci. USA 87,910~9112 18 Miller, C. (1991) Science 252, 1092-1096 19 Guy, H. R. and Conti, F. (1990) Trends Netrrosci. 13, 201-206 20 Ferry, D. R., Rombusch, M., GOB, A. and Glossmann, H. (1984) FEBS Letf. 169,112-167 21 Nakavama. H.. Taki. M.. Striessnie. 1.. Catterall, W. A. and .Kanaoka, Y. (%91) Proc. Nntl Acad. Sci. USA 88, 9203-9207 22 Galizzi, J. I’., Borsotto. M., Barhanin, J., Fosset, M. and Lazdunski, M. (1986) J. Biol. Chem. 261, 1393-1397 23 Takahashi, M., Seagar, M. J., Jones, J. F., Reber, B. F. and Catterall, W. A. (1987) Proc. N&l Acad. Sci. USA 84, 5478-5482 24 Vaahy, P. L. et al. (1987) J. Biol. Chem. 26z i4337-14342 25 Sharp, A. H., Imagawa, T., Leung, A. T. and Campbell, K. P. (1987) I. Biof. C/rem. 262,12309-12315 26 Hosev, M. M. et al. (1987) Biochem. Biophys. Res. Comnrnn. i47, 1137-1145 27 Regulla, S., Schneider, T., Nastainczyk, W., Meyer, H. E. and Hofmann, F. (1991) EMBO J. 10,45-49 28 Striessnig, J., Murphy, B. J. and Catterall, W. A. (1991) Proc. Natl Acad. Sci. USA 88,10769-10773 29 Kantrowitz, E. R. and Lipscomb, W. N. (1990) Treads Biochem. SC{. 15, 53-59 30 lohnson, L. N. and Barford. D. (1990) j. Biol. Cffem. 265, 2409-2412. ’ 31 Perutz, M. F. (1989) Qwrt. Rev. Biovhvs. . _ 22,139-236 32 Sprang, S., Goldsmith, E. and Fletterick, R. (1987) Science 237, 1012-1019 33 Hoshi, i., Zagotta, W. N. and Aldrich, R. W. (1990) Science 250,533-538 34 Hoshi, T., Zagotta, W. N. and Aldrich, R. W. (1991) Neuron 7, 547-556 35 Naito, K., McKenna, E., Schwartz, A. and Vaghy, I’. L. (1989) J. Biol. Chent. 264.21211-21214 36 Striessnig, J., Scheffauer, F., Mitterdorfer, J., Schirmer, M. and Glossmann, H. (1990) J. Biol. Clrem. 265, 363-370 D890: N-methyl methoxyverapamil Diazipine: 2-[4-(l-azi-2,2,2trifluoroethyl)benzoylamino]ethyl-2,6dimethvl-4-(2-trifluoromethvl)uhenvl-1.4dihydropyridine-3,5_dicarbo;);late ’ SDZ207180: (+)-lo-JJJ4-(4benzofurazanyi)-1,4_dihydro-2,6dimethyl-5-[(l-methylethoxy)carbonyl]-3pyridinylJcarbonyl]oxy]-N,N,N-trimethylI-decanaminium iodide

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