title: Opposite effects of a single IIIS5 mutation on phenylalkylamine and dihydropyridine interaction with L-type Ca2+ channels.
abstract: Replacement of L-type Ca(2+) channel alpha(1) subunit residue Thr-1066 in segment IIIS5 by a tyrosine residue conserved in the corresponding positions of non-L-type Ca(2+) channels eliminates high dihydropyridine sensitivity through a steric mechanism. To determine the effects of this mutation on phenylalkylamine interaction, we exploited the availability of Ca(v)1.2DHP(-/-) mice containing the T1066Y mutation. In contrast to dihydropyridines, increased protein-dependent binding of the phenylalkylamine (-)-[(3)H]devapamil occurred to Ca(v)1.2DHP(-/-) mouse brain microsomes. This effect could be attributed to an at least 2-fold increase in affinity as determined by saturation analysis and binding inhibition experiments. The latter also revealed a higher affinity for (-)-verapamil but not for (-)-gallopamil. The mutation caused a pronounced slowing of (-)-[(3)H]devapamil dissociation, indicating a stabilization of the drug-channel complex. The increased affinity of mutant channels was also evident in functional studies after heterologous expression of wild type and T1066Y channels in Xenopus laevis oocytes. 100 mum (-)-verapamil inhibited a significantly larger fraction of Ba(2+) inward current through mutant than through WT channels. Our results provide evidence that phenylalkylamines also interact with the IIIS5 helix and that the geometry of the IIIS5 helix affects the access and/or binding of different chemical classes of Ca(2+) channel blockers to their overlapping binding domains. Mutation of Thr-1066 to a non-L-type tyrosine residue can be exploited to differentially affect phenylalkylamine and dihydropyridine binding to L-type Ca(2+) channels.
authors.affiliation: Abteilung Pharmakologie und Toxikologie, Institut für Pharmazie, Universität Innsbruck, Peter-Mayrstrasse 1/I, A-6020 Innsbruck, Austria.
authors.forename: Irene G
authors.initials: IG
authors.lastname: Huber
authors.forename: Edwin
authors.initials: E
authors.lastname: Wappl-Kornherr
authors.forename: Martina J
authors.initials: MJ
authors.lastname: Sinnegger-Brauns
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Hoda
authors.forename: Doris
authors.initials: D
authors.lastname: Walter-Bastl
authors.forename: Jörg
authors.initials: J
authors.lastname: Striessnig
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000737:chemistry*; D015220:Calcium Channels / Q000737:chemistry*; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004095:Dihydropyridines / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005711:Gallopamil / Q000494:pharmacology; D006720:Homozygote; D017403:In Situ Hybridization; D017275:Isradipine / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013251:Steroid Isomerases / Q000235:genetics*; D014443:Tyrosine / Q000737:chemistry; D014700:Verapamil / Q000031:analogs & derivatives* / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D018076:DNA, Complementary; D004095:Dihydropyridines; C419385:L-type calcium channel alpha(1C); D018075:RNA, Complementary; D011994:Recombinant Proteins; D005711:Gallopamil; D014443:Tyrosine; C038806:1,4-dihydropyridine; D014700:Verapamil; C490380:Ebp protein, mouse; D013251:Steroid Isomerases; C055885:devapamil; D002118:Calcium; D017275:Isradipine