The single pore residue Asp(542) determines Ca2+ permeation and Mg2+ blockof the epithelial Ca2+ channel

The epithelial Ca2+ channel (ECaC), which was recently cloned from rabbit k idney, exhibits distinctive properties that support a facilitating role in transcellular Ca2+ (re)absorption, ECaC is structurally related to the fami ly of six transmembrane-spanning ion channels with a pore-forming region be tween 55 and S6, Using point mutants of the conserved negatively charged am ino acids present in the putative pore, we have identified a single asparta te residue that determines Ca2+ permeation of ECaC and modulation by extrac ellular Mg2+. Mutation of the aspartate residue, D542A, abolishes Ca2+ perm eation and Ca2+ dependent current decay as well as block by extracellular M g2+, whereas monovalent cations still permeate the mutant channel. Variatio n of the side chain length in mutations D542N, D542E, and D542M attenuated Ca2+ permeability and Ca2+-dependent current decay, Block of monovalent cur rents through ECaC by Mg2+ was decreased. Exchanging the aspartate residue for a positively charged amino acid, D542K, resulted in a nonfunctional cha nnel, Mutations of two neighboring negatively charged residues, i,e. Glu(53 5) and Asp(550), had only minor effects on Ca2+ permeation properties.