9 L-TYPE AMINO-ACID-RESIDUES CONFER FULL 1,4-DIHYDROPYRIDINE SENSITIVITY TO THE NEURONAL CALCIUM-CHANNEL ALPHA(1A) SUBUNIT - ROLE OF L-TYPEMET

Pharmacological modulation by 1,4-dihydropyridines is a central featur e of L-type calcium channels, Recently, eight L-type amino acid residu es in transmembrane segments IIIS5, IIIS6, and IVS6 of the calcium cha nnel alpha(1) subunit were identified to substantially contribute to 1 ,4-dihydropyridine sensitivity, To determine whether these eight L-typ e residues (Thr(1066), Gln(1070), Ile(1180), Ile(1183), Tyr(1490), Met (1491), Ile(1497), and Ile(1498), alpha(1C-a) numbering) are sufficien t 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-i nsensitive alpha(1A) subunit using site-directed mutagenesis, 1,4-Dihy dropyridine agonist and antagonist modulation of barium inward current s mediated by the mutant alpha(1A) subunits, coexpressed with alpha(2) delta and beta(1a) subunits in Xenopus laevis oocytes, was investigat ed with the two-microelectrode voltage clamp technique, The resulting mutant alpha(1A-DHPi) displayed low sensitivity for 1,4-dihydropyridin es. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type alpha(1) subunit previously cloned from Musca domestica body w all muscle led to the identification of Met(1188) (alpha(1C-a) numberi ng) as an additional critical constituent of the L-type 1,4-dihydropyr idine binding domain, The introduction of this residue into alpha(1A-D Hpi) restored full sensitivity for 1,4-dihydropyridines. It also trans ferred functional properties considered hallmarks of 1,4-dihydropyridi ne agonist and antagonist effects (i.e. stereoselectivity, voltage dep endence of drug modulation, and agonist-induced shift in the voltage-d ependence of activation), Our gain-of-function mutants provide an exce llent 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.