Two mechanistically distinct effects of dihydropyridine nifedipine on CaV1.2 L-type Ca²⁺ channels revealed by Timothy syndrome mutation

Abstract

Dihydropyridine Ca(2+) channel antagonists (DHPs) block Ca(V)1.2 L-type Ca(2+) channels (LTCCs) by stabilizing their voltage-dependent inactivation (VDI); however, it is still not clear how DHPs allosterically interact with the kinetically distinct (fast and slow) VDI. Thus, we analyzed the effect of a prototypical DHP, nifedipine on LTCCs with or without the Timothy syndrome mutation that resides in the I-II linker (L(I)-(II)) of Ca(V)1.2 subunits and impairs VDI. Whole-cell Ba(2+) currents mediated by rabbit Ca(V)1.2 with or without the Timothy mutation (G436R) (analogous to the human G406R mutation) were analyzed in the presence and absence of nifedipine. In the absence of nifedipine, the mutation significantly impaired fast closed- and open-state VDI (CSI and OSI) at -40 and 0 mV, respectively, but did not affect channels' kinetics at -100 mV. Nifedipine equipotently blocked these channels at -80 mV. In wild-type LTCCs, nifedipine promoted fast CSI and OSI at -40 and 0 mV and promoted or stabilized slow CSI at -40 and -100 mV, respectively. In LTCCs with the mutation, nifedipine resumed the impaired fast CSI and OSI at -40 and 0 mV, respectively, and had the same effect on slow CSI as in wild-type LTCCs. Therefore, nifedipine has two mechanistically distinct effects on LTCCs: the promotion of fast CSI/OSI caused by L(I-II) at potentials positive to the sub-threshold potential and the promotion or stabilization of slow CSI caused by different mechanisms at potentials negative to the sub-threshold potential.