Molecular determinants of permeation through the cation channel TRPM6

Abstract

TRPM6 and its closest relative TRPM7 are members of the Transient Receptor Potential Melastatin (TRPM) subfamily of cation channels and are known to be Mg 2+ permeable. By aligning the sequence of the putative TRPM6 pore with the pore sequences of the other subfamily members, we located in the loop between the fifth and the sixth transmembrane domain, a stretch of amino acids residues, 1028GEIDVC 1033, as the potential selectivity filter. Two negatively charged residues, E 1024 (conserved in TRPM6, TRPM7, TRPM1 and TRPM3) and D 1031 (conserved along the entire TRPM subfamily), were identified as important determinants of cation permeation through TRPM6, because neutralization of both residues into an alanine resulted in non-functional channels. Neutralization of E 1029 (conserved in TRPM6, TRPM7, TRPM4 and TRPM5) resulted in channels with increased conductance for Ba 2+ and Zn 2+, decreased ruthenium red sensitivity and larger pore diameter compared to wild-type TRPM6. Changing the residue I 1030 into methionine, resulted in channels with lower conductance for Ni 2+, decreased sensitivity to ruthenium red block and reduced pore diameter. Thus, these data demonstrate that amino acid residues E 1024, I 1030 and D 1031 are important for channel function and that subtle amino acid variation in the pore region accounts for TRPM6 permeation properties.