Nine L-type amino acid residues confer full 1,4-dihydropyridine sensitivity to the neuronal calcium channel alpha(1A) subunit - Role of L-type MET

Pharmacological modulation by 1,4-dihydropyridines is a central feature of L-type calcium channels, Recently, eight L-type amino acid residues in transmembrane segments IIIS5, IIIS6, and IVS6 of the calcium channel alpha(1) subunit were identified to substantially contribute to 1,4-dihydropyridine sensitivity, To determine whether these eight L-type residues (Thr(1066), Gln(1070), Ile(1180), Ile(1183), Tyr(1490), Met(1491), Ile(1497), and Ile(1498), alpha(1C-a) numbering) are sufficient to form a high affinity 1,4-dihydropyridine binding site in a non-L-type calcium channel, we transferred them to the 1,4-dihydropyridine-insensitive alpha(1A) subunit using site-directed mutagenesis, 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant alpha(1A) subunits, coexpressed with alpha(2) delta and beta(1a) subunits in Xenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique, The resulting mutant alpha(1A-DHPi) displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type alpha(1) subunit previously cloned from Musca domestica body wall muscle led to the identification of Met(1188) (alpha(1C-a) numbering) as an additional critical constituent of the L-type 1,4-dihydropyridine binding domain, The introduction of this residue into alpha(1A-DHpi) restored full sensitivity for 1,4-dihydropyridines. It also transferred functional properties considered hallmarks of 1,4-dihydropyridine agonist and antagonist effects (i.e. stereoselectivity, voltage dependence of drug modulation, and agonist-induced shift in the voltage-dependence of activation), Our gain-of-function mutants provide an excellent model for future studies of the structure-activity relationship of 1,4-dihydropyridines to obtain critical structural information for the development of drugs for neuronal, non-L-type calcium channels.