Effect of Mibefradil on Voltage-Dependent Gating and Kinetics of T-Type Ca Channels in Cortisol-Secreting Cells

We have studied the effect of the Ca antagonist mibefradil on low voltage-activated T-type Ca channels in whole-cell patch clamp recordings from bovine adrenal zona fasciculata (AZF) cells. AZF cells are distinctive in expressing only T-type Ca channels, allowing the mechanism of pharmacological agents to be explored without interference from other Ca channels. The inhibition of T-type Ca channels by mibefradil was voltageand use-dependent. When Ca currents were activated from holding potentials of 280 and 260 mV, mibefradil inhibited currents with IC50 values of 1.0 and 0.17 mM, respectively. When T-type Ca current (IT) was activated from a holding potential of 290 mV in the presence of 2 mM mibefradil, a single voltage step to 210 mV inhibited IT by 16.2% 6 2.9% (n 5 10). With subsequent voltage steps, applied at 10-s intervals, block reached a steady-state value of 51.9% 6 5.0% (n 5 5). Mibefradil (1 mM) produced a leftward shift of 5.7 mV (n 5 4) in the voltage-dependent steady-state availability curve such that Ttype Ca channels inactivated at more negative potentials, but this drug did not change the voltage-dependence of T channel opening. Mibefradil failed to alter the kinetics of T channel activation, inactivation, or deactivation, but markedly slowed T channel recovery following an inactivating prepulse. Mibefradil inhibited adrenocorticotropin-stimulated cortisol secretion from AZF cells with an IC50 value of 3.5 mM. These results show that mibefradil is a relatively potent antagonist of T-type Ca channels in cortisol-secreting cells. The enhanced potency of mibefradil with sustained or repetitive depolarizations, its shifting of the steady-state inactivation curve, and its slowing of recovery all indicate that this drug preferentially interacts with Ca channels in the open or inactivated state. The inhibition of cortisol secretion by mibefradil at concentrations similar to those that block IT is consistent with a requirement for these channels in corticosteroidogenesis. Mibefradil is a Ca channel antagonist that has been used clinically as an antihypertensive and antianginal agent (Noll and Lusher, 1998). Among organic Ca antagonists, mibefradil is distinctive because it preferentially blocks low voltage-activated Trather than L-type Ca channels (Mishra and Hermsmeyer, 1994b; Mehrke et al., 1994; Ertel and