A region of N-type Ca2+ channel critical for blockade by the dihydropyridine amlodipine

Abstract 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 α 1B α 2/ δ 1 β 1a combination, the N-type channel formed a high affinity binding site for [ 3 H]amlodipine (K d , 3.08 nM) and was profoundly blocked by amlodipine (IC 50 , 2.7 μM at − 60 mV). By contrast, R-type (α 1E α 2/ δ 1 β 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 α 1 subunit, which are involved in dihydropyridine binding in L-type channels, the two amino acid residues Lys 1287 (corresponding to Met 1295 in α1B) and Phe 1699 (corresponding to Leu 1697 in α 1B ) were unique in α 1E . An amino acid substitution of Lys1287Met in IIIS5 of α 1E conferred a high affinity binding site for amlodipine (K d , 13.1 nM) and a sensitivity to amlodipine (IC 50 , 11.3 μM). In N-type channel, reversely, an amino acid substitution of Met1295Lys in IIIS5 of α 1B deprived a high affinity binding site for amlodipine and reduced the channel blockade by amlodipine (IC 50 , 29.6 μM). The results indicate that Met 1295 in the region IIIS5 of α 1B is critical for amlodipine to efficiently bind and block the N-type Ca 2+ channel.