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 [³H]-mibefradil and a series of mibefradil analogues to L-type Ca²⁺ channels in different tissues. 2. [³H]-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 (and a series of analogues) partially inhibited (+)-[³H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca²⁺ channels and α1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. 4. [³H]-Mibefradil also labelled 3 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 μM 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.