High affinity interaction of mibefradil with voltage-gated calcium and sodium channels.

1 Mibefradil is a novel Ca2+ antagonist which blocks both high-voltage activated and low voltage-activated Ca2+ channels. Although L-type Ca2+ channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [H-3]-mibefradil and a series of mibefradil analogues to L-type Ca2+ channels in different tissues. 2 [H-3]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca2+ channels (K-D of 2.5 +/- 0.4 nM, B-max = 56.4 +/- 2.3 pmol mg(-1) of protein). 3 Mibefradil land a series of analogues) partially inhibited (+)-[H-3]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca2+ channels and alpha 1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. 4 [H-3]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca2+ and Na+-channel blockers. 5 Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na+-channels as confirmed in binding (apparent K-i values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na+-channel current by 1 mu M mibefradil in GH3 cells). 6 Our data demonstrate that mibefradil binds to voltage-gated L-type Ca2+ channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na+-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca2+-/Na+-channel blockers.