Effect of mibefradil on voltage-dependent gating and kinetics of T-type Ca2+ channels in cortisol-secreting cells

We have studied the effect of the Ca2+ antagonist mibefradil on low voltage -activated T-type Ca2+ channels in whole-cell patch clamp recordings from b ovine adrenal zona fasciculata (AZF) cells. AZF cells are distinctive in ex pressing only T-type Ca2+ channels, allowing the mechanism of pharmacologic al agents to be explored without interference from other Ca2+ channels. The inhibition of T-type Ca2+ channels by mibefradil was voltage- and use-depe ndent. When Ca2+ currents were activated from holding potentials of -80 and -60 mV, mibefradil inhibited currents with IC50 values of 1.0 and 0.17 mu M, respectively. When T-type Ca2+ current (IT) was activated from a holding potential of -90 mV in the presence of 2 mu M mibefradil, a single voltage step to -10 mV inhibited I-T by 16.2% +/- 2.9% (n = 10). With subsequent v oltage steps, applied at 10-s intervals, block reached a steady-state value of 51.9% +/- 5.0% (n = 5). Mibefradil (1 mu M) produced a leftward shift o f 5.7 mV (n = 4) in the voltage- dependent steady-state availability curve such that T-type Ca2+ channels inactivated at more negative potentials, but this drug did not change the voltage-dependence of T channel opening. Mibe fradil failed to alter the kinetics of T channel activation, inactivation, or deactivation, but markedly slowed T channel recovery following an inacti vating prepulse. Mibefradil inhibited adrenocorticotropin-stimulated cortis ol secretion from AZF cells with an IC50 value of 3.5 mu M. These results s how that mibefradil is a relatively potent antagonist of T-type Ca2+ channe ls in cortisol-secreting cells. The enhanced potency of mibefradil with sus tained or repetitive depolarizations, its shifting of the steady-state inac tivation curve, and its slowing of recovery all indicate that this drug pre ferentially interacts with Ca2+ channels in the open or inactivated state. The inhibition of cortisol secretion by mibefradil at concentrations simila r to those that block IT is consistent with a requirement for these channel s in corticosteroidogenesis.