Opposite Effects of a Single IIIS5 Mutation on Phenylalkylamine and Dihydropyridine Interaction with L-type Ca super(2+) Channels

• Published in: The Journal of biological chemistry Vol. 279; no. 53; pp. 55211 - 55217
• Main Authors: Huber, Irene G, Wappl-Kornherr, Edwin, Sinnegger-Brauns, Martina J, Hoda, Jean-Charles, Walter-Bastl, Doris, Striessnig, Joerg
• Format: Journal Article
• Language: English
• Published: 31.12.2004
• Subjects: Xenopus laevis
• ISSN: 0021-9258; 1083-351X

Replacement of L-type Ca super(2+) channel alpha sub(1) subunit residue Thr-1066 in segment IIIS5 by a tyrosine residue conserved in the corresponding positions of non-L-type Ca super(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 sub(v)1.2DHP super(-/-) mice containing the T1066Y mutation. In contrast to dihydropyridines, increased protein-dependent binding of the phenylalkylamine (-)- [ super(3)H]devapamil occurred to Ca sub(v)1.2DHP super(-/-) 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 (-)-[ super(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 mu M (-)-verapamil inhibited a significantly larger fraction of Ba super(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 super(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 super(2+) channels.