Direct inhibition of voltage-dependent Ca²⁺ fluxes by ethanol and higher alcohols in rabbit T-tubule membranes

The effects of ethanol and higher alcohols on ⁴⁵Ca²⁺ fluxes, mediated by voltage-dependent Ca²⁺ channels (VDCCs), were investigated in inside-out transverse (T)-tubule membrane vesicles from rabbit skeletal muscle. ⁴⁵Ca²⁺ effluxes were induced by membrane potentials generated via establishing K⁺ gradients across the vesicles, and were significantly inhibited by the inorganic Ca²⁺ channel blocker La³⁺ (1 mM) and the Ca²⁺ channel antagonist nifedipine (1-10 μM). Ethanol, in the concentration range of 100-400 mM, caused a significant suppression of depolarization-induced ⁴⁵Ca²⁺ fluxes. Ethanol also functionally modulated the effect of nifedipine (1-10 μM) and the Ca²⁺ channel agonist Bay K 8644 (1 μM) on Ca²⁺ effluxes. Pretreatment with pertussis toxin (5 μg/ml) or phorbol 12-myrstate 13-acetate (PMA, 50 nM) did not affect the ethanol inhibition of ⁴⁵Ca²⁺ fluxes. Further experiments with alcohols revealed that butanol, hexanol, octanol and decanol also significantly inhibited ⁴⁵Ca²⁺ effluxes. However, undecanol and dodecanol did not cause any significant change on ⁴⁵Ca²⁺ fluxes, indicating that the effects of alcohols on ⁴⁵Ca²⁺ effluxes exhibit a cut-off phenomenon. In radioligand binding studies, it was found that at the concentrations used in flux studies, alcohols did not alter the characteristics of the specific binding of [³H]PN 200-110 to T-tubule membranes. Results indicate that ethanol directly inhibits the function of voltage-dependent Ca²⁺ channels without modulating the specific binding of Ca²⁺ channel ligands of the dihydropyridine class, and that this inhibition is independent of intracellular Ca²⁺ levels.