Molecular Determinants of High Affinity Phenylalkylamine Block of l-type Calcium Channels in Transmembrane Segment IIIS6 and the Pore Region of the α1Subunit

Abstract

Recent studies of the phenylalkylamine binding site in the α1C subunit of l-type Ca2+ channels have revealed three amino acid residues in transmembrane segment IVS6 that are critical for high affinity block and are unique to l-type channels. We have extended this analysis of the phenylalkylamine binding site to amino acid residues in transmembrane segment IIIS6 and the pore region. Twenty-two consecutive amino acid residues in segment IIIS6 were mutated to alanine and the conserved Glu residues in the pore region of each homologous domain were mutated to Gln. Mutant channels were expressed in tsA-201 cells along with the β1b and α2δ auxiliary subunits. Assay for block of Ba2+ current by (−)-D888 at −60 mV revealed that mutation of five amino acid residues in segment IIIS6 and the pore region that are conserved between l-type and non-l-type channels (Tyr1152, Phe1164, Val1165, Glu1118, and Glu1419) and one l-type-specific amino acid (Ile1153) decreased affinity for (−)-D888 from 10–20-fold. Combination of the four mutations in segment IIIS6 increased the IC50 for block by (−)-D888 to approximately 9 μm, similar to the affinity of non-l-type Ca2+ channels for this drug. These results indicate that there are important determinants of phenylalkylamine binding in both the S6 segments and the pore regions of domains III and IV, some of which are conserved across the different classes of voltage-gated Ca2+ channels. A model of the phenylalkylamine receptor site at the interface between domains III and IV of the α1 subunit is presented. Recent studies of the phenylalkylamine binding site in the α1C subunit of l-type Ca2+ channels have revealed three amino acid residues in transmembrane segment IVS6 that are critical for high affinity block and are unique to l-type channels. We have extended this analysis of the phenylalkylamine binding site to amino acid residues in transmembrane segment IIIS6 and the pore region. Twenty-two consecutive amino acid residues in segment IIIS6 were mutated to alanine and the conserved Glu residues in the pore region of each homologous domain were mutated to Gln. Mutant channels were expressed in tsA-201 cells along with the β1b and α2δ auxiliary subunits. Assay for block of Ba2+ current by (−)-D888 at −60 mV revealed that mutation of five amino acid residues in segment IIIS6 and the pore region that are conserved between l-type and non-l-type channels (Tyr1152, Phe1164, Val1165, Glu1118, and Glu1419) and one l-type-specific amino acid (Ile1153) decreased affinity for (−)-D888 from 10–20-fold. Combination of the four mutations in segment IIIS6 increased the IC50 for block by (−)-D888 to approximately 9 μm, similar to the affinity of non-l-type Ca2+ channels for this drug. These results indicate that there are important determinants of phenylalkylamine binding in both the S6 segments and the pore regions of domains III and IV, some of which are conserved across the different classes of voltage-gated Ca2+ channels. A model of the phenylalkylamine receptor site at the interface between domains III and IV of the α1 subunit is presented. l-type Ca2+ channels are found in many excitable cell types, including muscle, neuronal, and endocrine cells, where they initiate Ca2+-dependent responses such as contraction and secretion (reviewed in Refs. 1Snutch T.P. Reiner P.B. Curr. Opin. Neurobiol. 1992; 2: 247-253Crossref PubMed Scopus (251) Google Scholar and 2Catterall W.A. Curr. Opin. Cell Biol. 1994; 6: 607-615Crossref PubMed Scopus (47) Google Scholar). The pore forming α1 subunits of voltage-gated Ca2+channels consist of four homologous domains (I-IV), each containing six putative transmembrane segments (S1-S6) (3Tanabe T. Takeshima H. Mikami A. Flockerzi V. Takahashi H. Kangawa K. Kojima M. Matsuo H. Hirose T. Numa S. Nature. 1987; 328: 313-318Crossref PubMed Scopus (978) Google Scholar). The role ofl-type channels in initiating muscle contraction within the cardiovascular system has made them important therapeutic targets for the treatment of hypertension, angina pectoris, and cardiac arrhythmia (4Fleckenstein A. Fleckenstein-Grun G. Eur. Heart J. 1980; 1: 15-21Crossref PubMed Scopus (51) Google Scholar). Three major classes of l-type Ca2+ channel blockers are currently in clinical use, dihydropyridines, benz(othi)azepines, and phenylalkylamines. Drugs from all three classes bind to the pore-forming α1 subunit ofl-type Ca2+ channels in a manner that suggests that their binding sites are closely linked (5Gould R.J. Murphy K.M. Snyder S.H. Cold Spring Harbor Symp. Quant. Biol. 1983; 48: 355-362Crossref PubMed Google Scholar, 6Glossmann, H., Ferry, D. R., Goll, A., Rombusch, M. (1984)J. Cardiovasc. Pharmacol. , 6, (Suppl.) 508–521.Google Scholar). Recently, much progress has been made toward the characterization of Ca2+antagonist receptor sites at the molecular level (for review see Ref.7Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar). Photoaffinity labeling studies and studies utilizing chimeric and mutant channels have indicated that both transmembrane segments IIIS6 and IVS6 are involved in forming the dihydropyridine (8Striessnig J. Murphy B.J. Catterall W.A. Proc. Natl. Acac. Sci. U. S. A. 1991; 88: 10769-10773Crossref PubMed Scopus (123) Google Scholar, 9Grabner M. Wang Z. Hering S. Striessnig J. Glossmann H. Neuron. 1996; 16: 207-218Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 10Peterson B.Z. Tanada T.N. Catterall W.A. J. Biol. Chem. 1996; 271: 5293-5296Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) and benz(othi)azepine (11Kraus R. Reichl B. Kimball S.D. Grabner M. Murphy B.J. Catterall W.A. Striessnig J. J. Biol. Chem. 1996; 271: 20113-20118Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) binding sites, and that dihydropyridine binding involves transmembrane segment IIIS5 as well (12Mitterdorfer J. Wang Z. Sinnegger M.J. Hering S. Striessnig J. Grabner M. Glossmann H. J. Biol. Chem. 1996; 271: 30330-30335Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In contrast, such studies of the phenylalkylamine binding site, to date, have indicated the involvement of only transmembrane segment IVS6. The high affinity photolabel LU49888 was found to derivatize transmembrane segment IVS6 of the skeletal muscle l-type channel exclusively (13Striessnig J. Glossmann H. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9108-9112Crossref PubMed Scopus (140) Google Scholar). In subsequent studies with mutant channels, three amino acid residues in IVS6, which are unique tol-type channels, have been found to be critical for high affinity phenylalkylamine block (14Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Schuster A. Lacinova L. Klugbauer N. Ito H. Birnbaumer L. Hoffmann F. EMBO J. 1996; 15: 2365-2370Crossref PubMed Scopus (115) Google Scholar). Simultaneous mutation of these three residues, Tyr1463, Ala1467, and Ile1470, resulted in channels that were blocked by the high affinity phenylalkylamine (−)-D888 with an affinity similar to that of non-l-type channels. Transfer of the l-type IVS6 sequence in chimeric Ca2+ channels was sufficient to confer l-type sensitivity to (−)-D888 in a non-l-type channel (16Doring F. Degtiar V.E. Grabner M. Striessnig J. Hering S. Glossmann H. J. Biol. Chem. 1996; 271: 11745-11749Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Despite the studies implicating only segment IVS6 in phenylalkylamine binding, several lines of evidence suggest that IIIS6 might also be involved in the phenylalkylamine receptor site. The allosteric interactions among all three major classes of l-type Ca2+ channel blockers suggests that the binding sites for these drugs are near each other but not identical. Because evidence from both photolabeling studies and analysis of mutant channels has implicated domain IIIS6 in the binding of both dihydropyridines and benzothiazepines, it is important to examine the role of individual amino acids in domain IIIS6 in block of l-type channels by phenylalkylamines. We have individually mutated 21 amino acids in IIIS6 to Ala and another (Ala1157) to Pro and analyzed the mutant channels for sensitivity to the high affinity phenylalkylamine (−)-D888. We report here that four amino acid residues in transmembrane domain IIIS6 are required for high affinity block ofl-type Ca2+ channels by (−)-D888. The phenylalkylamines are thought to block ion channels by occluding the ion-conducting pore and thereby preventing cation permeation (17Wit A.L. Cranefield P.F. Circ. Res. 1974; 35: 413-425Crossref PubMed Scopus (237) Google Scholar, 18Hescheler J. Pelzer D. Trube G. Trautwein W. Pflügers Arch. Eur. J. Physiol. 1982; 393: 287-291Crossref PubMed Scopus (176) Google Scholar, 19Pelzer D. Trautwein W. McDonald T.F. Pflügers Archi.v. 1982; 394: 97-105Crossref PubMed Scopus (79) Google Scholar). Moreover, at physiological pH, phenylalkylamines are predominantly positively charged due to protonation of the tertiary amino group. In view of these observations, we mutated four conserved Glu residues known to contribute to the ion-selective pore ofl-type Ca2+ channels, Glu363, Glu709, Glu1118, and Glu1419 (20Yang J. Ellinor P.T. Sather W.A. Zhang J.F. Tsien R.W. Nature. 1993; 366: 109-110Crossref PubMed Scopus (532) Google Scholar), to Gln and screened the resulting mutant channels for sensitivity to (−)-D888. We report here that Glu1118 and Glu1419 in the putative pore-lining segments of homologous domains III and IV, respectively, are also involved in high affinity block of l-type Ca2+ channels by phenylalkylamines. All mutations were constructed using oligonucleotide-directed mutagenesis as described previously (21Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4905) Google Scholar). The IIIS6, E1118Q, and E1419Q mutations were inserted into full-length α1 subunit constructs in the expression vector Zem229 (Dr. Eileen Mulvihill, Zymogenetics Corp., Seattle) using the 1.5-kilobase SpeI/DraIII fragment and the 272-base pair DraIII/DraIII fragment in a three-way ligation. The E363Q mutation was inserted into the full-length α1 subunit construct using the 1.4-kilobaseNgoMI/BglII fragment. The E709Q mutation was inserted into the full-length α1 subunit construct using the 1.3-kilobase SgrAI/SpeI fragment. All mutations were confirmed by cDNA sequencing. tsA201 cells, a subclone of the human embryonic kidney cell line HEK293 that expresses SV40 T antigen (a gift of Dr. Robert Dubridge, Cell Genesis, Foster City, CA), were maintained in monolayer culture in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Life Technologies, Inc.), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), and incubated at in 10% and mutant channel subunits T.P. Neuron. 1991; Full Text PDF PubMed Scopus Google Scholar) were expressed with β1b M. J. S.D. 1991; PubMed Scopus Google Scholar) and R. A. A. PubMed Scopus Google Scholar) channel subunits and antigen in tsA-201 cells from by as described B. R. 1994; 15: Scholar). were by labeling with to (−)-D888 was to cells in the cell by the of of a to a The and to with and to with All were at were these were from and to an of were using a and at were using have been for using an Ca2+ channel α1C subunit T.P. Neuron. 1991; Full Text PDF PubMed Scopus Google Scholar) was expressed in tsA-201 cells B. R. 1994; 15: Scholar) with the β1b M. J. S.D. 1991; PubMed Scopus Google Scholar) and R. A. A. PubMed Scopus Google Scholar) subunits. Ba2+ the Ca2+ channels were blocked by a of (−)-D888 the Ba2+ current by approximately The block by (−)-D888 was and within (14Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). of block of Ba2+ by a of of (−)-D888 an IC50 of The putative transmembrane segment IIIS6 of the α1C subunit of l-type Ca2+channels amino acid the amino acid residues to this transmembrane are different in the (14Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) subunits of Ca2+ channels the role of the individual amino acid residues in the IIIS6 segment of α1C in high affinity block by we mutated to Pro as in Ca2+ channels mutated the other amino acids in IIIS6 to and screened the mutant channels for sensitivity to (−)-D888. Ala was for it has in in the of M. Zhang 1993; PubMed Scopus Google Scholar) and is to the and of the amino acid in each in this putative All of the mutant α1 subunits Ca2+ channels in tsA-201 cells of these mutations the of block by as for in In contrast, three of these amino acid mutations in the IC50 for block of Ba2+ by (−)-D888. the mutation a in the of (−)-D888 required for block of the Ba2+ current and approximately a to of the for (−)-D888 The IC50 for block of Ba2+ the and all of the mutant α1C subunits are as a in the 21 amino acid mutations to Ala only and in the (−)-D888 block In to the other mutations the IC50 for (−)-D888 approximately to a important role of the amino acid in this in affinity for phenylalkylamines. the of a at this we mutated to of The which only a an in the of (−)-D888 required for block of and approximately an to in the for (−)-D888 IC50 results suggest that a of four amino acid residues in segment IIIS6 contribute to of the high affinity receptor site for (−)-D888 these four amino acid residues are determinants of high affinity binding of mutation of of them the IC50 for block of the l-type Ca2+ channels to a near μm, the Ca2+ channels (14Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The mutation an in the IC50 for block by (−)-D888 the of (−)-D888 in the of were required to block of Ba2+ of the block of Ba2+ by a of of (−)-D888 at an IC50 of of in these an level of block with mutant and with the other that the in IC50 in the for The in of binding of (−)-D888 by each mutation be from the to the the with (−)-D888 binding, the and the the was the in binding in the was only of the of the by the individual that the to the binding by these four amino acid residues are not examine the of the of the IIIS6 mutations we their and with of Ca2+channels containing were similar for and for the mutant α1C subunits affinity for (−)-D888 A and with Ba2+ at to mV in each analysis of revealed but and These were not due to in the from forming the cell were at and were that the of was different for the the of channel was not different in the mutant with that the of the mutations were in the The IIIS6 mutations also the of the mutant channels, which is a of their ion of the mutant was only from mV both mutations were approximately mV of and were also from the of mutant was These results are with the that the IIIS6 segment to the of the pore of Ca2+ channels, and mutations of amino acid residues in this segment the of ion In to their of channel phenylalkylamines Ca2+ channel to in the that block by these is at where is T. R. J. Pharmacol. Google Scholar, T.F. Pelzer D. Trautwein W. J. Physiol. PubMed Scopus Google Scholar, B.D. G.H. Scheuer T. Catterall W.A. Pharmacol. 1996; Google Scholar). mutations that of Ca2+ channels binding and block by phenylalkylamines such all of were at a of −60 of l-type Ca2+ channels, in by mutations not affinity for (−)-D888. we have the of of the mutant Ca2+ channels in of residues Phe1164, and for and with of the from not the of but of the from that in IIIS6 resulted in a of approximately mV in for the mutant are approximately mV examine the between in the of and the affinity for block by (−)-D888 we the IC50 for the IIIS6 their from mV to and with IC50 is mutant has the as but increased other have increased decreased of but in and have different but in IC50 is at the in these and of IC50 is the in (−)-D888 affinity be to in the of channel in the the other the of the for the in affinity by mutation mutant has by the and also has the affinity for (−)-D888. at −60 mV for this mutant contribute to increased affinity for (−)-D888. The α1 subunits of voltage-gated Ca2+ channels four conserved between the and S6 transmembrane segments of each homologous domain that the which the channel The high of voltage-gated Ca2+channels for and for Ca2+ other is by four Glu residues, one in each homologous that are conserved across all Ca2+ channels (20Yang J. Ellinor P.T. Sather W.A. Zhang J.F. Tsien R.W. Nature. 1993; 366: 109-110Crossref PubMed Scopus (532) Google Scholar, S. G. A. A. G. A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). Because phenylalkylamines are thought to block ion channels by binding in the pore and the amino of (−)-D888 is positively charged at physiological and with the pore Glu residues an we mutated each of these Glu residues individually to Gln to the that the have at physiological and the affinity for block by (−)-D888 in the four The mutant channels E363Q and E709Q mutations in domains and respectively, were both blocked by (−)-D888 with IC50 that were not different from that of the channel A and the mutant channels and E1419Q in domains III and IV, respectively, both in the IC50 for (−)-D888 block and The mutant approximately a in the of block by (−)-D888 IC50 The E1419Q mutant approximately a in the of block by (−)-D888 and IC50 These results indicate that the Glu residues in domains III and IV with but in domains and the (−)-D888 mutation of the pore Glu residues other of channel The for these four that the of channel was not different from the of the mutant E363Q was approximately mV to the of mutant was approximately mV with of the mutations in domains and IV, E709Q and were not different from The of the pore mutations the of also In mutant the for was not different from for of E709Q and E1419Q were to by approximately and with the mutations in segment IIIS6, there was between the of the mutations IC50 for (−)-D888 for with the that these mutations the binding of (−)-D888 in we have analyzed the of 21 amino acid in the transmembrane segment IIIS6 to block by (−)-D888. the amino acid residues mutated in this only four the IC50 for (−)-D888. of these four amino acids is unique to l-type channels the are conserved across all of voltage-gated Ca2+ channels The involvement of conserved amino acid residues in segment IIIS6 in high affinity (−)-D888 binding in to the results of in segment IVS6 where the only three amino acids required for (−)-D888 binding were unique tol-type channels (14Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Schuster A. Lacinova L. Klugbauer N. Ito H. Birnbaumer L. Hoffmann F. EMBO J. 1996; 15: 2365-2370Crossref PubMed Scopus (115) Google Scholar, F. Degtiar V.E. Grabner M. Striessnig J. Hering S. Glossmann H. J. Biol. Chem. 1996; 271: 11745-11749Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). results that conserved amino acid residues in the ofl-type drugs and the of analysis of all amino acid residues in putative binding sites in to chimeric that only The amino acid mutation in this region with the was the of for in which only a was from the channel is similar to B.D. G.H. Scheuer T. Catterall W.A. Pharmacol. 1996; Google Scholar) that mutation of a in IVS6 to a in sensitivity to (−)-D888. Simultaneous mutation of both and to resulted in a channel with an approximately in IC50 for (−)-D888 and H. B. D. M. R. T. and W. A. The of these which are suggests a between them and the a the of is not for the affinity phenylalkylamines and which an B.D. G.H. Scheuer T. Catterall W.A. Pharmacol. 1996; Google Scholar). results the that the of the mutations of amino acid residues Phe1164, and binding of (−)-D888 results from of the interactions of the of these residues with the drug. The of mutations of these residues are mutations of residues to Ala have block by (−)-D888. is between the of these mutations of Ca2+ channels and their affinity for that the mutations not their by allosteric is that these four amino acid residues with (−)-D888 it is to receptor site l-type Ca2+ channels with high previously for amino acid residues in IVS6 that are required for high affinity phenylalkylamine block of l-type Ca2+ channels (14Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), it is that the IIIS6 amino acid residues that (−)-D888 block also into the ion-conducting the IVS6 Tyr1463, mutation of Phe1164, and to Ala resulted in in we for (14Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), these are to be due to in channel to the which is the cation in the mutations of Phe1164, and the of the ion-conducting are thought to bind in the pore of Ca2+ channels (17Wit A.L. Cranefield P.F. Circ. Res. 1974; 35: 413-425Crossref PubMed Scopus (237) Google Scholar, 18Hescheler J. Pelzer D. Trube G. Trautwein W. Pflügers Arch. Eur. J. Physiol. 1982; 393: 287-291Crossref PubMed Scopus (176) Google Scholar, 19Pelzer D. Trautwein W. McDonald T.F. Pflügers Archi.v. 1982; 394: 97-105Crossref PubMed Scopus (79) Google Scholar), and results that amino acid residues in transmembrane segments IIIS6 and IVS6 that are involved in high affinity binding of (−)-D888 are also involved in the ion of the pore G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar and this We the of four conserved Glu residues, Glu363, Glu709, Glu1118, and in the pore region for these amino acid into the pore to the Ca2+ binding that Ca2+ to voltage-gated Ca2+channels (20Yang J. Ellinor P.T. Sather W.A. Zhang J.F. Tsien R.W. Nature. 1993; 366: 109-110Crossref PubMed Scopus (532) Google Scholar, S. G. A. A. G. A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). these amino acid are charged at physiological and in interactions with positively phenylalkylamine The of the of mutations of these four pore Glu residues is with the of domains III and IV to be the site of binding of phenylalkylamines. of Glu1118 in domain III and domain IV major in the affinity for mutations of in domain and in domain The decreased affinity of and E1419Q for (−)-D888 was not by in the of the pore mutant with the in in affinity for the drug. it is that the decreased affinity for (−)-D888 by and E1419Q is due to the for these mutations among the four pore Glu residues and their of channel that they with the interactions with the positively charged amino group. is with studies that phenylalkylamines block ion channels in their positively J. Pelzer D. Trube G. Trautwein W. Pflügers Arch. Eur. J. Physiol. 1982; 393: 287-291Crossref PubMed Scopus (176) Google Scholar, J. Physiol. 1995; PubMed Scopus Google Scholar) and with the evidence that phenylalkylamines bind within the pore of the Ca2+ The results of this and studies (14Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Schuster A. Lacinova L. Klugbauer N. Ito H. Birnbaumer L. Hoffmann F. EMBO J. 1996; 15: 2365-2370Crossref PubMed Scopus (115) Google Scholar, F. Degtiar V.E. Grabner M. Striessnig J. Hering S. Glossmann H. J. Biol. Chem. 1996; 271: 11745-11749Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) suggest that the receptor site for (−)-D888 is of at four IIIS6, the in the IVS6, and the in the The critical role of amino acid residues from both IIIS6 and IVS6 in (−)-D888 binding and block suggest that these transmembrane domains are to a of the of the ion-conducting pore results a interface of phenylalkylamine binding and as previously for dihydropyridines W.A. Striessnig J. Pharmacol. Sci. 1992; Full Text PDF PubMed Scopus Google Scholar). The residues are in of amino acids each in IIIS6, but these four residues not in the in consecutive of the as the in IVS6 (14Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). the from of many in of known all four of these residues to contribute to a binding site for phenylalkylamines in the pore of the We that the in IIIS6 and the in IVS6 to a that (−)-D888 in the pore and the interactions between the pore Glu residues Glu1118 and Glu1419 and the tertiary amino of (−)-D888 be of to other phenylalkylamines of affinity with these of the high affinity phenylalkylamine receptor site. we have in the B.Z. Johnson B.D. G.H. M.J. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), mutation of amino acid residues in transmembrane segments IIIS6 and IVS6 the affinity of the l-type Ca2+ channel for the dihydropyridine as by The results of mutations in IIIS6 and IVS6 block by (−)-D888 and binding of are in B. is that the binding sites for these are mutations at three residues in in IIIS6 and IVS6 (Tyr1152, and both binding and (−)-D888 mutations at other mutation of Phe1164, Val1165, Ala1467, and block by (−)-D888 but not mutations of five amino acids in the region of IIIS6 and as well as four amino acids in IVS6 and each binding but not (−)-D888 We suggest that the phenylalkylamines and bind to different of the IIIS6 and IVS6 transmembrane segments and in some bind to of the amino acid In this the allosteric interactions between phenylalkylamines and by the of a the of an