A region of N-type Ca2+ 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 Ca2+ 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 [3H]amlodipine. When expressed as the α1Bα2/δ1β1a combination, the N-type channel formed a high affinity binding site for [3H]amlodipine (Kd, 3.08 nM) and was profoundly blocked by amlodipine (IC50, 2.7 μM at − 60 mV). By contrast, R-type (α1Eα2/δ1β1a) channels did not possess a high affinity binding site for [3H]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 α1 subunit, which are involved in dihydropyridine binding in L-type channels, the two amino acid residues Lys1287 (corresponding to Met1295 in α1B) and Phe1699 (corresponding to Leu1697 in α1B) were unique in α1E. An amino acid substitution of Lys1287Met in IIIS5 of α1E conferred a high affinity binding site for amlodipine (Kd, 13.1 nM) and a sensitivity to amlodipine (IC50, 11.3 μM). In N-type channel, reversely, an amino acid substitution of Met1295Lys in IIIS5 of α1B deprived a high affinity binding site for amlodipine and reduced the channel blockade by amlodipine (IC50, 29.6 μM). The results indicate that Met1295 in the region IIIS5 of α1B is critical for amlodipine to efficiently bind and block the N-type Ca2+ channel.