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J. Biol. Chem., Vol. 282, Issue 35, 25817-25830, August 31, 2007

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Molecular Determinants of Mg²⁺ and Ca²⁺ Permeability and pH Sensitivity in TRPM6 and TRPM7^*

Mingjiang Li¹², Jianyang Du¹, Jianmin Jiang³, William Ratzan, Li-Ting Su, Loren W. Runnels, and Lixia Yue⁴

From the Center for Cardiology and Cardiovascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06030 and the Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg²⁺ and Ca²⁺ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg²⁺ and Ca²⁺ permeability and pH sensitivity remains unknown. Here we have created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the permeability of the channel and its regulation by pH. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I-V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8- and 12-fold larger than TRPM7, respectively. The binding affinity of Ca²⁺ and Mg²⁺ was decreased by 50- to 140-fold in E1052Q and E1047Q, respectively. Ca²⁺ and Mg²⁺ currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca²⁺ and Mg²⁺ permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E1047Q, indicating that E1047 is critical to Ca²⁺ and Mg²⁺ permeability of TRPM7, and its pH sensitivity. Mutation of the corresponding residues in the pore of TRPM6, E1024Q and E1029Q, produced nearly identical changes to the channel properties of TRPM6. Our results indicate that these two glutamates are key determinants of both channels' divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/pathological functions of TRPM6 and TRPM7, and will extend our understanding of the pore structures of TRPM channels.

Received for publication, September 20, 2006 , and in revised form, June 26, 2007.

^* This work was supported by American Heart Association Grant 0335124N and National Institutes of Health Grant HL078960 (to L. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Fig. S1.

¹ Both authors contributed equally to this work.

² Present address: Dept. of Cardiology, Renmin Hospital of Wuhan University, Peoples Republic of China.

³ Present address: Dept. of Pharmacology and Toxicology, Sun Yat-Sen University, Peoples Republic of China.

⁴ To whom correspondence should be addressed. Tel.: 860-679-3869; Fax: 860-679-1426; E-mail: lyue{at}uchc.edu.

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