The high affinity phenylalkylamine(-)D888 blocks ion currents through L-type Ca channels containing the subunit with an apparent Kof 50 nM, but N-type Ca channels in the pheochromocytoma cell line PC12 are blocked with a 100-fold higher K value of 5 µM. L-type Ca channels containing 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 . Ca 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 Ca 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 -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.

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