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

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. [3H]‐Mibefradil labelled a single class of high affinity sites on skeletal muscle L‐type Ca2+ channels (KD of 2.5±0.4 nM, Bmax=56.4±2.3 pmol mg−1 of protein). 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. [3H]‐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. Mibefradil and its analogue RO40‐6040 had high affinity for neuronal voltage‐gated Na+‐channels as confirmed in binding (apparent Ki 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). 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. British Journal of Pharmacology (2000) 130, 669–677; doi:10.1038/sj.bjp.0703352