Molecular determinants of high affinity phenylalkylamine block of L-type calcium channels.

The high affinity phenylalkylamine (-)D888 blocks ion currents through L-type Ca2+ channels containing the alpha 1C subunit with an apparent Kd of 50 nM, but N-type Ca2+ channels in the pheochromocytoma cell line PC12 are blocked with a 100-fold higher Kd value of 5 microM. L-type Ca2+ channels containing alpha 1C subunits with the site-directed mutations Y1463A, A1467S, or I1470A in the putative transmembrane segment S6 in domain IV (IVS6) were 6-12 times less sensitive to block by (-)D888 than control alpha 1C. Ca2+ channels containing paired combinations of these mutations were even less sensitive to block by (-)D888 than the single mutants, and channels containing all three mutations were > 100 times less sensitive to (-)D888 block, similar to N-type Ca2+ channels. In addition, the Y1463A mutant and all combination mutants including the Y1463A mutation had altered ion selectivity, suggesting that Tyr-1463 faces the pore and is involved in ion permeation. Since these three critical amino acid residues are aligned on the same face of the putative IVS6 alpha-helix, we propose that they contribute to a receptor site in the pore that confers a high affinity block of L-type channels by (-)D888.