Voltage-dependent blockade of diverse types of voltage-gated Ca²⁺ channels expressed in Xenopus oocytes by the Ca²⁺ channel antagonist mibefradil (Ro 40-5967)

Four different types of Ca²⁺ channel α1 subunits, representing the major classes of voltage-gated Ca²⁺ channels, were individually coexpressed along with α2/δ and β2b subunits in Xenopus oocytes. These subunits (and the encoded channel types and major tissues of origin) included α1C (L- type, cardiac), α1B (N-type, central nervous system), α1A (P/Q-type, central nervous system), and α1E (most likely R-type, central nervous system). Divalent cation currents through these channels (5 mM Ba²⁺) were evaluated with the two-microelectrode voltage-clamp technique. The expressed channels were compared with regard to their responses to a structurally novel, nondihydropyridine compound, mibefradil (Ro 40-5967). In the micromolar concentration range, this drug exerted clear inhibitory effects on each of the four channel types, reducing divalent cation current at all test potentials, with the non-L-type channels being more sensitive to inhibition than the L-type channels under fixed experimental conditions. For all channel types, mibefradil was a much more effective inhibitor at more depolarized holding potentials, suggesting tighter binding of the drug to the inactivated state than to the resting state. The difference in apparent affinities of resting and inactivated states of the channels, calculated based on a modulated receptor hypothesis, was 30-70-fold. In addition, the time course of decay of Ca²⁺ channel current was accelerated in the presence of drug, consistent with open channel block. The effect of increasing stimulation frequency was tested for L-type channels and was found to greatly enhance the degree of inhibition by mibefradil, consistent with promotion of block by channel opening and inactivation. Allowing for state-dependent interactions, the drug concentrations found to block L-, N-, Q-, and R-type channels by 50% are at least 10-fold higher than half-blocking levels previously reported for T-type channels in vascular smooth muscle cells under similar experimental conditions. This may help explain the ability of the drug to spare working myocardium (strongly negative resting potential, dominance of L-type channels in their resting state) while reducing contraction in blood vessels (presumably involving T-type channels or partially inactivated L-type channels). Thus, mibefradil is a new addition to the family of nonselective organic Ca²⁺ channel inhibitors, as exemplified by bepridil and fluspirilene, and may prove useful as an experimental tool for studying diverse physiological events initiated by Ca²⁺ influx. It complements classes of drugs with relatively selective effects on L-type channels, as exemplified by nifedipine and diltiazem.