Abstract

Mibefradil is a Ca²⁺ channel antagonist that inhibits both T-type and high-voltage-activated Ca²⁺ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC₅₀ of NNC 55-0396 to block recombinant α₁G T-type channels in human embryonic kidney 293 cells was ∼7 μM, whereas 100 μM NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca²⁺ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca²⁺ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca²⁺ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca²⁺ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca²⁺ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca²⁺ channels, thus rendering it more selective to T-type Ca²⁺ channels.