MUTATIONS IN THE EF-HAND MOTIF IMPAIR THE INACTIVATION OF BARIUM CURRENTS OF THE CARDIAC ALPHA(1C) CHANNEL

Calcium-dependent inactivation has been described as a negative feedba ck mechanism for regulating voltage-dependent calcium influx in cardia c cells. Most recent evidence oints to the C-terminus of the alpha(1C) subunit, with its EF-hand binding motif, as being critical in this pr ocess. The EF-hand binding motif is mostly conserved between the C-ter mini of six of the seven alpha(1) subunit Ca2+ channel genes. The role of E1537 in the C-terminus of the alpha(1C) calcium channel inactivat ion was investigated here after expression in Xenopus laevis oocytes. Whole-cell currents were measured in the presence of 10 mM Ba2+ or 10 mM Ca2+ after intracellular injection of ,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid. Against all expectations, our results show ed a significant reduction in the rate of voltage-dependent inactivati on as measured in Ba2+ solutions for all E1537 mutants, whereas calciu m-dependent inactivation appeared unscathed. Replacing the negatively charged glutamate residue by neutral glutamine, glycine, serine, or al anine significantly reduced the rate of Ba2+-dependent inactivation by 1.5-fold (glutamine) to 3.5-fold (alanine). The overall rate of macro scopic inactivation measured in Ca2+ solutions was also reduced, altho ugh a careful examination of the distribution of the fast and slow tim e constants suggests that only the slow time constant was significantl y reduced in the mutant channels. The fast time constant, the hallmark of Ca2+-dependent inactivation, remained remarkably constant among wi ld-type and mutant channels. Moreover, inactivation of E1537A channels , in both Ca2+ and Ba2+ solutions, appeared to decrease with membrane depolarization, whereas inactivation of wild-type channels became fast er with positive voltages. All together, our results showed that E1537 mutations impaired voltage-dependent inactivation and suggest that th e proximal part of the C-terminus may play a role in voltage-dependent inactivation in L-type alpha(1C) channels.