Distinct effects of mutations in transmembrane segment IVS6 on block of L-type calcium channels by structurally similar phenylalkylamines.

The phenylalkylamines (-)-D888, verapamil, and D600, cause voltage- and use-dependent block of L-type Ca2+ channels and differ from each other only in the number of methoxy groups on each of their two terminal phenyl rings. To study the effects of mutations in the phenylalkylamine receptor site on block by these drugs, wild-type and mutant Ca2+ channels were transiently expressed in the tsA-201 clone of human embryonic kidney 293 cells. The combined mutations Y1463A, A1467S, and I1470A (mutant YAI) in transmembrane segment S6 of domain IV of the alpha 1c subunit disrupted block by all three phenylalkylamines. Surprisingly, although this mutation reduced both resting block at -60 mV and depolarized block at +10 mV by (-)-D888, resting and depolarized block by verapamil and D600 were relatively unaffected. In contrast, for all three drugs, use-dependent block during repetitive stimulations was sharply reduced, and the rate of recovery from depolarized block was accelerated for YAI channels. Thus, the effects of the YAI mutation on apparent affinity were specific to (-)-D888, whereas effects on the kinetics of block were observed for all three drugs. Additional experiments with substitution of phenylalanine for Y1463 suggested that (-)-D888 affinity is specifically sensitive to removal of the hydroxyl group of Y1463, whereas effects on the kinetics of block by all three phenylalkylamines require larger molecular changes, perhaps related to residue size and hydrophobicity. Analysis of the data using a state-dependent model of drug block suggests that these kinetic differences are caused by both changes in drug access to the receptor site and affinity for binding to the inactivated state of the channel. The different effects of the YAI mutations on the actions of (-)-D888, verapamil, and D600 indicate that these residues interact differently with these closely related drugs.