Motif III S5 of L-type calcium channels is involved in the dihydropyridine binding site. A combined radioligand binding and electrophysiological study.

The alpha1 subunit of L-type voltage-dependent Ca2+ channels (alpha1C) has been shown to harbor high affinity binding sites for the Ca2+ channel dihydropyridine (DHP) modulators. It has been suggested by a number of investigators that the binding site may be composed of III S6 and IV S6. Evidence with chimeric channels indicated the possible involvement of III S5 in DHP binding. Site-directed mutations were introduced in motif III S5 region of the alpha1C, changing the amino acids to their counterparts in the DHP-insensitive alpha1A channel. The mutant channels were expressed both in HEK 293 cells and in Xenopus oocytes. Equilibrium binding and electrophysiological studies showed that the Thr1006 to Tyr substitution produced a mutant channel with at least 1000-fold decreased affinity in [3H](+)isopropyl-4-(2,1, 3-benzoxadiazol-4-yl)-1,4-dihydro-(2, 6-dimethyl-5-methoxycarbonyl)pyridine-3-carboxylate (PN200-110, isradipine) binding and in sensitivity of R(-)-4(2,1, 3-benzoxadiazol-4-yl)-1,4-dihydro-2, 6-dimethyl-5-nitro-3-pyridincarboxylic acid isopropylester (R202-791) in terms of inhibition of current through the L-type voltage-dependent Ca2+ channels. Replacing Gln1010 with Met resulted in more than a 10-fold decrease in binding affinity for [3H](+)PN200-110 and in the potency of channel modulation by S202-791. Four additional mutations in this region also lead to a slight but statistically significant increase of KD values for [3H](+)PN200-110 binding. The binding and electrophysiological results show that certain residues of the transmembrane segment III S5 are important in contributing to the DHP binding "pocket" and are critical for DHP binding and for its calcium channel effect.