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

The structural determinants of mibefradil inhibition were analyzed using wi ld-type and inactivation-modified Ca(v)1.2 (alpha 1C) and Ca(v)2.3 (alpha 1 E) channels. Mibefradil inhibition of peak Ba2+ currents was dose- and volt age-dependent. An increase of holding potentials from -80 to -100 mV signif icantly shifted dose-response curves toward higher mibefradil concentration s, namely from a concentration of 108 +/- 21 muM (n = 7) to 288 +/- 17 muM (n = 3) for inhibition of half of the Ca(v)1.2 currents (IC50) and from IC5 0 = 8 +/- 2 Lm (n = 9) to 33 7 Lm (n = 4) for Cav2.3 currents. In the prese nce of mibefradil, Ca(v)1.2 and Ca(v)2.3 experienced significant use-depend ent inhibition (0.1 to I Hz) and slower recovery from inactivation suggesti ng mibefradil could promote transition(s) to an absorbing inactivated state . In order to investigate the relationship between inactivation and drug se nsitivity, mibefradil inhibition was studied in inactivation-altered Ca(v)1 .2 and Ca(v)2.3 mutants. Mibefradil significantly delayed the onset of chan nel recovery from inactivation in CEEE (Repeat I + part of the I-H linker f rom 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 P-subunit binding motif) c hannels. Mibefradil inhibited the faster inactivating chimera EC(ISI-6)EEE with an IC50 = 7 +/- 1 muM (n = 3), whereas the slower inactivating chimera s EC(AID)EEE and CEEE were, respectively, inhibited with IC50 = 41 +/- 5 mu M (n = 4) and IC50 = 68 +/- 9 muM (n = 5). Dose-response curves were superi mposable for the faster EC(ISI-6)EEE and Ca(v)2.3, whereas intermediate-ina ctivating channel kinetics (CEEE, Ca(v)1.2 E462R, and Ca(v)1.2 E462K) were inhibited by similar concentrations of mibefradil with IC50 approximate to 55-75 muM. The slower Ca(v)1.2 wild-type and Ca(v)1.2 Q473K channels respon ded to higher doses of mibefradil with IC50 = 100-120 pm. Mibefradil was al so found to significantly speed up the inactivation kinetics of slower chan nels (Ca(v)1.2, CEEE) with little effect on the inactivation kinetics of fa ster-inactivating channels (Ca(v)2.3). A open-channel block model for mibef radil interaction with high-voltage-activated Ca2+ channels is discussed an d shown to qualitatively account for our observations. Hence. our data agre e reasonably well with a "receptor guarded mechanism" where fast inactivati on kinetics efficiently trap mibefradil into the channel.