Blockade of Ba super(2+) current through human alpha 1E 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) Ca super(2+) currents. At least some of these effects appear to involve direct blocking actions on Ca super(2+) channels. Thus, direct investigation of the effects of various steroids on cloned Ca super(2+) channel variants may lead to the development of potent and selective small-molecular weight Ca super(2+) channel blockers. Here we examine the effects of two steroids on a cloned human alpha 1E Ca super(2+) channel both with and without a beta 3 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. (+)-ACN can block 100% of Ba super(2+) current in HEK cells arising either from the alpha 1E subunit (IC sub(50) similar to 10 mu M) or the alpha 1E beta 3 combination (IC sub(50) similar to 5 mu M), while (+)-ECN maximally blocks only about 80% of the alpha 1E (10 mu M) or alpha 1E beta 3 (16 mu M) current. Blockade by (+)-ACN exhibits several differences from blockade by (+)-ECN. (+)-ACN increases the apparent rate of onset of inactivation, particularly for the alpha 1E variant, slows recovery from inactivation, and more profoundly shifts the voltage-dependence of current availability for both alpha 1E and alpha 1E beta 3 variants than does (+)-ECN. Although the complexity of the normal inactivation kinetics of alpha 1E 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.