Nine L-type amino acid residues confer full 1,4-dihydropyridine sensitivity to the neuronal calcium channel alpha1A subunit. Role of L-type Met1188

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 alpha1 subunit were identified to substantially contribute to 1,4-dihydropyridine sensitivity. To determine whether these eight L-type residues (Thr1066, Gln1070, Ile1180, Ile1183, Tyr1490, Met1491, Ile1497, and Ile1498; alpha1C-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 alpha1A subunit using site-directed mutagenesis. 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant alpha1A subunits, coexpressed with alpha2delta and beta1a subunits in Xenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique. The resulting mutant alpha1A-DHPi displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type alpha1 subunit previously cloned from Musca domestica body wall muscle led to the identification of Met1188 (alpha1C-a numbering) as an additional critical constituent of the L-type 1,4-dihydropyridine binding domain. The introduction of this residue into alpha1A-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.