Molecular determinants of sensitivity and conductivity of human TRPM7 to Mg2+ and Ca2+
Molecular determinants of sensitivity and conductivity of human TRPM7 to Mg2+ and Ca2+
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DOI：
10.4161/chan.2.4.6695
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发表时间：
2008-07-01
期刊：
影响因子：
3.3
通讯作者：
Okada, Yasunobu
中科院分区：
文献类型：
--
作者：
Numata, Tomohiro;Okada, Yasunobu
已知胞外Mg2+和Ca2+可以渗透TRPM7，同时通过单价阳离子阻断其渗透。在本研究中，我们研究了人类TRPM7对这些二价阳离子的电导率和敏感性的分子基础。胞外酸化至pH 4.0显著降低了Mg2+和Ca2+对Cs+电流的阻断作用，降低了它们的结合亲和力：它们的IC50值分别增加了510倍和447倍。我们检测了在人类TRPM7的假设成孔区域中和四个带负电荷的氨基酸残基（glu1047、glu1052、Asp-1054和Asp-1059）的效果。将Glu-1047突变为丙氨酸（E1047A）导致无功能通道，而将任何其他残基突变都会导致功能性表达通道。通过D1054A和E1052A的Cs+电流对二价阳离子阻断的敏感性较低；Mg2+的IC50值分别提高5.5倍和3.9倍，Ca2+的IC50值分别提高10.5倍和6.7倍。D1059A对Mg2+（1.7倍）和Ca2+（3-9倍）的敏感性也有显著降低，尽管与D1054A和E1052A相比，降低幅度较小。D1054A突变在很大程度上消除了由Mg2+和Ca2+传递的内向电流。在E1052A和D1059A突变体中，向内的Mg2+和Ca2+电流相当大，但明显减少。由此得出结论，在人TRPM7中，(1)位于孔选择性过滤器最窄部分附近的Asp-1054和asp -1052可能为Mg2+和Ca2+提供结合位点，(2)Asp-1054是Mg2+和Ca2+电导率的重要决定因素，(3)glug -1052和Asp-1059促进二价阳离子的传导。
It is known that extracellular Mg2+ and Ca2+ 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 Mg2+ and Ca2+ on the Cs+ currents, decreasing their binding affinities: their IC50 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 IC50 values were increased 5.5- and 3.9-fold, respectively, for Mg2+ and 10.5- and 6.7-fold, respectively, for Ca2+. D1059A also had a significant reduction, though less marked compared to the reductions seen for D1054A and E1052A, in sensitivity to Mg2+ (1.7-fold) and Ca2+ (3-9-fold). The D1054A mutation largely abolished inward currents conveyed by Mg2+ and Ca2+. In the E1052A and D1059A mutants, inward Mg2+ and Ca2+ 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 Mg2+ and Ca2+, (2) Asp-1054 is an essential determinant of Mg2+ and Ca2+ conductivitv, and (3) Glu-1052 and Asp-1059 facilitate the conduction of divalent cations.