Feedback inhibition of Ca 2+ channels by Ca 2+ depends on a short sequence of the C terminus that does not include the Ca 2+ -binding function of a motif with similarity to Ca 2+ -binding domains

Abstract

α_1C- and α_1E-based Ca²⁺ channels differ in that the former are inhibited by Ca²⁺ entering through its pore, while the latter are not. It has been proposed on the basis of analysis of α_1E/α_1C chimeras that the molecular determinants responsible for Ca²⁺ inhibition involve both a conserved Ca²⁺-binding motif (EF hand) plus additional sequences located C-terminal to the EF hand. Through construction of similar α_1E/α_1C chimeras, we transferred Ca²⁺ inhibition from α_1C to α_1E by replacing a 134-aa segment of α_1E with the homologous 142-aa segment of α_1C. This segment is located immediately after the proposed Ca²⁺-binding EF hand motif. Replacement of the α_1C EF hand with the corresponding EF hand of α_1E did not interfere with inhibition of α_1C by Ca²⁺, and a triple mutant of α_1C, α_1C[D1535A,E1537A,D1546A], that disrupts the potential Ca²⁺-coordinating ability of the EF hand continued to be inhibited by Ca²⁺. These results indicate that a small portion of the α_1C C terminus is essential for inhibition by Ca²⁺ and place the Ca²⁺-binding site anywhere in α_1C, with the exception of its EF hand-like motif.