Mutations in the EF-Hand Motif Impair the Inactivation of Barium Currents of the Cardiac α1C Channel

Abstract

Calcium-dependent inactivation has been described as a negative feedback mechanism for regulating voltage-dependent calcium influx in cardiac cells. Most recent evidence points to the C-terminus of the α 1C subunit, with its EF-hand binding motif, as being critical in this process. The EF-hand binding motif is mostly conserved between the C-termini of six of the seven α 1 subunit Ca 2+ channel genes. The role of E1537 in the C-terminus of the α 1C calcium channel inactivation was investigated here after expression in Xenopus laevis oocytes. Whole-cell currents were measured in the presence of 10 mM Ba 2+ or 10 mM Ca 2+ after intracellular injection of 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid. Against all expectations, our results showed a significant reduction in the rate of voltage-dependent inactivation as measured in Ba 2+ solutions for all E1537 mutants, whereas calcium-dependent inactivation appeared unscathed. Replacing the negatively charged glutamate residue by neutral glutamine, glycine, serine, or alanine significantly reduced the rate of Ba 2+-dependent inactivation by 1.5-fold (glutamine) to 3.5-fold (alanine). The overall rate of macroscopic inactivation measured in Ca 2+ solutions was also reduced, although a careful examination of the distribution of the fast and slow time constants suggests that only the slow time constant was significantly reduced in the mutant channels. The fast time constant, the hallmark of Ca 2+-dependent inactivation, remained remarkably constant among wild-type and mutant channels. Moreover, inactivation of E1537A channels, in both Ca 2+ and Ba 2+ solutions, appeared to decrease with membrane depolarization, whereas inactivation of wild-type channels became faster with positive voltages. All together, our results showed that E1537 mutations impaired voltage-dependent inactivation and suggest that the proximal part of the C-terminus may play a role in voltage-dependent inactivation in L-type α 1C channels.