Molecular Determinants of the Differential Modulation of Cav1.2 and Cav1.3 by Nifedipine and FPL 64176

Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca 2+ channels, respectively, modulate Ca v 1.2 more potently than Ca v 1.3. To identify potential strategies for developing subtype-selective inhibitors, we investigated the role of divergent amino acid residues in transmembrane domains IIIS5 and the extracellular IIIS5-3P loop region in modulation of these channels by nifedipine and FPL. Insertion of the extracellular IIIS5-3P loop from Ca v 1.2 into Ca v 1.3 (Ca v 1.3+) reduced the IC 50 of nifedipine from 289 to 101 nM, and substitution of S1100 with an A residue, as in Ca v 1.2, accounted for this difference. Substituting M1030 in IIIS5 to V in Ca v 1.3+ (Ca v 1.3+V) further reduced the IC 50 of nifedipine to 42 nM. FPL increased current amplitude with an EC 50 of 854 nM in Ca v 1.3, 103 nM in Ca v 1.2, and 99 nM in Ca v 1.3+V. In contrast to nifedipine block, substitution of M1030 to V in Ca v 1.3 had no effect on potency of FPL potentiation of current amplitude, but slowed deactivation in the presence and absence of 10 μ M FPL. FPL had no effect on deactivation of Ca v 1.3/dihydropyridine-insensitive (DHPi), a channel with very low sensitivity to nifedipine block (IC 50 ∼93 μ M), but did shift the voltage-dependence of activation by ∼-10 mV. We conclude that the M/V variation in IIIS5 and the S/A variation in the IIIS5-3P loop of Ca v 1.2 and Ca v 1.3 largely determine the difference in nifedipine potency between these two channels, but the difference in FPL potency is determined by divergent amino acids in the IIIS5-3P loop.