Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University College of Pharmacy, West Lafayette, Indiana.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University College of Pharmacy, West Lafayette, Indiana
Mol Pharmacol. 2018 Sep;94(3):973-983. doi: 10.1124/mol.118.112441. Epub 2018 Jul 6.
Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca channels, respectively, modulate Ca1.2 more potently than Ca1.3. 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 Ca1.2 into Ca1.3 (Ca1.3+) reduced the IC of nifedipine from 289 to 101 nM, and substitution of S1100 with an A residue, as in Ca1.2, accounted for this difference. Substituting M1030 in IIIS5 to V in Ca1.3+ (Ca1.3+V) further reduced the IC of nifedipine to 42 nM. FPL increased current amplitude with an EC of 854 nM in Ca1.3, 103 nM in Ca1.2, and 99 nM in Ca1.3+V. In contrast to nifedipine block, substitution of M1030 to V in Ca1.3 had no effect on potency of FPL potentiation of current amplitude, but slowed deactivation in the presence and absence of 10 M FPL. FPL had no effect on deactivation of Ca1.3/dihydropyridine-insensitive (DHPi), a channel with very low sensitivity to nifedipine block (IC ∼93 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 Ca1.2 and Ca1.3 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.
硝苯地平和 FPL 64176（FPL）分别阻断和增强 L 型电压门控钙通道，对 Ca1.2 的调制作用强于 Ca1.3。为了确定开发亚型选择性抑制剂的潜在策略，我们研究了跨膜结构域 IIIS5 和细胞外 IIIS5-3P 环区域中发散氨基酸残基在硝苯地平和 FPL 对这些通道的调制中的作用。将 Ca1.2 的细胞外 IIIS5-3P 环插入 Ca1.3（Ca1.3+）中，将硝苯地平的 IC 从 289 降低到 101 nM，而 S1100 被 A 残基取代，如 Ca1.2 中的那样，解释了这种差异。将 IIIS5 中的 M1030 替换为 Ca1.3+中的 V（Ca1.3+V）进一步将硝苯地平的 IC 降低至 42 nM。FPL 在 Ca1.3 中增加电流幅度的 EC 为 854 nM，在 Ca1.2 中为 103 nM，在 Ca1.3+V 中为 99 nM。与硝苯地平阻断相反，在 Ca1.3 中，M1030 取代 V 对 FPL 增强电流幅度的效力没有影响，但在存在和不存在 10 M FPL 的情况下，使失活速度变慢。FPL 对 Ca1.3/二氢吡啶不敏感（DHPi）的失活没有影响，该通道对硝苯地平阻断的敏感性非常低（IC∼93 M），但确实将激活的电压依赖性移动了约-10 mV。我们得出结论，Ca1.2 和 Ca1.3 中 IIIS5 的 MV 变化和 IIIS5-3P 环中的 SA 变化在这两个通道之间的硝苯地平效力差异中起主要作用，但 FPL 效力的差异取决于 IIIS5-3P 环中的发散氨基酸。
