State-dependent inhibition of inactivation-deficient Ca(V)1.2 and Ca(V)2.3 channels by mibefradil.

The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified Ca(V)1.2 (alpha1C) and Ca(V)2.3 (alpha1E) channels. Mibefradil inhibition of peak Ba2+ currents was dose- and voltage-dependent. An increase of holding potentials from -80 to -100 mV significantly shifted dose-response curves toward higher mibefradil concentrations, namely from a concentration of 108 +/- 21 microm (n = 7) to 288 +/- 17 microm (n = 3) for inhibition of half of the Ca(V)1.2 currents (IC(50)) and from IC(50) = 8 +/- 2 microm (n = 9) to 33 +/- 7 microm (n = 4) for Ca(V)2.3 currents. In the presence of mibefradil, Ca(V)1.2 and Ca(V)2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation and drug sensitivity, mibefradil inhibition was studied in inactivation-altered Ca(V)1.2 and Ca(V)2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of the I-II linker from Ca(V)1.2 in the Ca(V)2.3 host channel), in EC(AID)EEE (part of the I-II linker from Ca(V)1.2 in the Ca(V)2.3 host channel) as well as in Ca(V)1.2 E462R, and Ca(V)2.3 R378E (point mutation in the beta-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC(50) = 7 +/- 1 microm (n = 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC(50) = 41 +/- 5 microm (n = 4) and IC(50) = 68 +/- 9 microm (n = 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and Ca(V)2.3, whereas intermediate-inactivating channel kinetics (CEEE, Ca(V)1.2 E462R, and Ca(V)1.2 E462K) were inhibited by similar concentrations of mibefradil with IC(50) approximately 55-75 microm. The slower Ca(V)1.2 wild-type and Ca(V)1.2 Q473K channels responded to higher doses of mibefradil with IC(50) approximately 100-120 microm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (Ca(V)1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (Ca(V)2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca2+ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with a "receptor guarded mechanism" where fast inactivation kinetics efficiently trap mibefradil into the channel.