The single pore residue Asp542 determines Ca2+ permeation and Mg2+ block of the epithelial Ca2+ channel

Abstract

The epithelial Ca2+ channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca2+ (re)absorption. ECaC is structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region between S5 and S6. Using point mutants of the conserved negatively charged amino acids present in the putative pore, we have identified a single aspartate residue that determines Ca2+ permeation of ECaC and modulation by extracellular Mg2+. Mutation of the aspartate residue, D542A, abolishes Ca2+ permeation and Ca2+-dependent current decay as well as block by extracellular Mg2+, whereas monovalent cations still permeate the mutant channel. Variation of the side chain length in mutations D542N, D542E, and D542M attenuated Ca2+ permeability and Ca2+-dependent current decay. Block of monovalent currents through ECaC by Mg2+ was decreased. Exchanging the aspartate residue for a positively charged amino acid, D542K, resulted in a nonfunctional channel. Mutations of two neighboring negatively charged residues, i.e. Glu535 and Asp550, had only minor effects on Ca2+ permeation properties.