| Mibefradil (Ro 40-5967) is a tetralol derivative once marketed as Posicor (Roche) to treat hypertension. Despite significant therapeutic benefits, mibefradil was withdrawn from the market because of a fatal drug interaction involving inhibition of a cytochrome P450 enzyme. Mibefradil, however, remains an intriguing compound and an important prototype drug for structural studies of its targets, which include the two classes of Ca2+ channels found in the cardiovascular system, i.e. L- and T-type. Several reports have also demonstrated that mibefradil blocks other classes of ion channels including K+ channels, Cl- channels, and Na+ channels, raising the possibility that miberadil's hemodynamic effects are due to its interaction with multiple targets and also points to a potentially common mechanism of action across targets. My thesis work detailed mibefradil's action on Na+ and T-type Ca2+ channels. Mibefradil blocked both classes of channels in a tonic and use-dependent manner with an affinity that was strongly dependent on channel state. Charge measurements demonstrated that channels continued to gate in the presence of saturating drug concentrations suggesting that current inhibition is not mediated via immobilization of the channel's voltage sensors, but rather by occlusion of the channel pore. Mibefradil, however, did not exhibit a preference for the open state nor on states preferentially filled from the open state, i.e. a pre-open closed state or the fast-inactivated state. Higher affinity was observed during high frequency stimulation, under conditions of modest depolarization, and when channels were selectively recruited to populate slow-inactivated conformations. Furthermore, mutations of an asparagine in Domain I, S6 near the inner pore enhance slow inactivation and increased affinity 10--20 fold. These data support a model in which mibefradil interacts with longer-lived inactivated conformations that reveal a high affinity binding site located near the inner pore. Insights from this work may not only facilitate future drug design, but the dose range and novel state-dependent profile of mibefradil block also make it well suited for structure/function studies of its channel targets, for identification of common drug binding motifs, and for determination of how drug binding is affected by conformational changes during channel gating. |
