A region of N-type Ca(2+) channel critical for blockade by the dihydropyridine amlodipine

Amlodipine, a dihydropyridine derivative, has been shown to block not only L-type but also N-type Ca(2+) channels. Aiming to understand the mechanism underlying such a selective blockade by amlodipine, the interaction of amlodipine with N-type channels was investigated using the Xenopus oocyte expression system together with the two-microelectrode voltage-clamp technique and the binding assay for [(3)H]amlodipine. When expressed as the alpha(1B)alpha(2/)delta(1)beta(1a) combination, the N-type channel formed a high affinity binding site for [(3)H]amlodipine (K(d), 3.08nM) and was profoundly blocked by amlodipine (IC(50), 2.7 microM at -60mV). By contrast, R-type (alpha(1E)alpha(2/)delta(1)beta(1a)) channels did not possess a high affinity binding site for [(3)H]amlodipine and their channel activities were not influenced by amlodipine. In comparison of amino acid sequences in the transmembrane regions IIIS5, IIIS6 and IVS6 of the alpha(1) subunit, which are involved in dihydropyridine binding in L-type channels, the two amino acid residues Lys(1287) (corresponding to Met(1295) in alpha1B) and Phe(1699) (corresponding to Leu(1697) in alpha(1B)) were unique in alpha(1E). An amino acid substitution of Lys1287Met in IIIS5 of alpha(1E) conferred a high affinity binding site for amlodipine (K(d), 13.1nM) and a sensitivity to amlodipine (IC(50), 11.3 microM). In N-type channel, reversely, an amino acid substitution of Met1295Lys in IIIS5 of alpha(1B) deprived a high affinity binding site for amlodipine and reduced the channel blockade by amlodipine (IC(50), 29.6 microM). The results indicate that Met(1295) in the region IIIS5 of alpha(1B) is critical for amlodipine to efficiently bind and block the N-type Ca(2+) channel.