Molecular determinants of sensitivity and conductivity of human TRPM7 to Mg2+ and Ca2+.
It is known that extracellular Mg(2+) and Ca(2+) can permeate TRPM7 and at the same time block the permeation by monovalent cations. In the present study, we examined the molecular basis for the conductivity and sensitivity of human TRPM7 to these divalent cations. Extracellular acidification to pH 4.0 markedly reduced the blocking effects of Mg(2+) and Ca(2+) on the Cs(+) currents, decreasing their binding affinities: their IC(50) values increased 510- and 447-fold, respectively. We examined the effects of neutralizing each of four negatively charged amino acid residues, Glu-1047, Glu-1052, Asp-1054 and Asp-1059, within the putative pore-forming region of human TRPM7. Mutating Glu-1047 to alanine (E1047A) resulted in non-functional channels, whereas mutating any of the other residues resulted in functionally expressed channels. Cs(+) currents through D1054A and E1052A were less sensitive to block by divalent cations; the IC(50) values were increased 5.5- and 3.9-fold, respectively, for Mg(2+) and 10.5- and 6.7-fold, respectively, for Ca(2+). D1059A also had a significant reduction, though less marked compared to the reductions seen for D1054A and E1052A, in sensitivity to Mg(2+) (1.7-fold) and Ca(2+) (3.9-fold). The D1054A mutation largely abolished inward currents conveyed by Mg(2+) and Ca(2+). In the E1052A and D1059A mutants, inward Mg(2+) and Ca(2+) currents were sizable but significantly diminished. Thus, it is concluded that in human TRPM7, (1) both Asp-1054 and Glu-1052, which are located near the narrowest portion in the pore's selectivity filter, may provide the binding sites for Mg(2+) and Ca(2+), (2) Asp-1054 is an essential determinant of Mg(2+)and Ca(2+) conductivity, and (3) Glu-1052 and Asp-1059 facilitate the conduction of divalent cations.
Numata
Tomohiro
T
Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.
Okada
Yasunobu
Y
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry*; D002412:Cations; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D008274:Magnesium / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D017346:Protein Serine-Threonine Kinases; D017386:Sequence Homology, Amino Acid; D050053:TRPM Cation Channels / Q000378:metabolism*
D002412:Cations; D050053:TRPM Cation Channels; D017346:Protein Serine-Threonine Kinases; C486583:TRPM7 protein, human; D008274:Magnesium; D002118:Calcium