Blockade of Ba2+ current through human alpha1E channels by two steroid analogs, (+)-ACN and (+)-ECN

Previous work suggests that different neuroactive steroids may exhibit some selectivity in their blocking effects on different high-voltage activated (HVA) Ca2+ currents. At least some of these effects appear to involve direct blocking actions on Ca2+ channels. Thus, direct investigation of the effects of various steroids on cloned Ca2+ channel variants may lead to the development of potent and selective small-molecular weight Ca2+ channel blockers. Here we examine the effects of two steroids on a cloned human alpha1E Ca2+ channel both with and without a beta3 subunit, when expressed in HEK293 cells. One compound, (+)-ACN, has been previously shown to block N-, Q-, and R-subtypes of HVA current without affecting L- and P-type current. The second compound, (+)-ECN, weakly blocks total HVA current in hippocampal neurons. (+)-ECN differs from (+)-ACN in lacking effects on GABA receptors, but shares with (+)-ACN an ability to partially inhibit T current in DRG neurons (Todorovic, S.M., Prakriya, M., Nakashima, Y.M. et al., 1998. Enantioselective blockade of T-type Ca2+ current in adult rat sensory neurons by a steroid lacking GABA-mimetic activity. Mol. Pharmacol. 54, 918-927). (+)-ACN can block 100% of Ba2+ current in HEK cells arising either from the alpha1E subunit (IC50 approximate to 10 microM) or the alpha1Ebeta3 combination (IC50 approximate to 5 microM), while (+)-ECN maximally blocks only about 80% of the alpha1E (10 microM) or alpha1Ebeta3 (16 microM) current. Blockade by (+)-ACN exhibits several differences from blockade by (+)-ECN. (+)-ACN increases the apparent rate of onset of inactivation, particularly for the alpha1E variant, slows recovery from inactivation, and more profoundly shifts the voltage-dependence of current availability for both alpha1E and alpha1Ebeta3 variants than does (+)-ECN. Although the complexity of the normal inactivation kinetics of alpha1E variants makes interpretation of the (+)-ACN-induced kinetic alterations difficult, the results suggest that the two steroids are to some extent acting by distinct mechanisms, and perhaps at different sites.