Abstract 955: Molecular Mechanisms of Ca 2+ and Mg 2+ Permeability and pH Sensitivity of the Bi-functional Channel Kinase TRPM7

Abstract

The channel-kinase TRPM7 have recently been discovered to play important roles in Mg 2+ and Ca 2+ homeostasis, which is critical to both human health and cell viability. TRPM7 is highly expressed in both cardiac myocytes and fibroblasts. We recently found that both heterologous expressed TRPM7 currents and endogenous TRPM7-like currents in the heart are sensitive to changes of extracellular pH. Acidic pH significantly potentiates inward current of TRPM7. Given the importance of TRPM7 in Ca 2+ and Mg 2+ homeostasis and potential pathological role of TRPM7 under acidosis conditions, this study was designed to elucidate the molecular basis underlying TRPM7’s unique Mg 2+ and Ca 2+ permeability and pH sensitivity. We created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the channel’s divalent permeability and pH sensitivity. 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 that of TRPM7, respectively. The binding affinity of Ca 2+ and Mg 2+ was decreased by 50-to 140-fold in E1052Q and E1047Q, respectively. Ca 2+ and Mg 2+ currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca 2+ and Mg 2+ permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E 1047Q and largely diminished in E1052Q, indicating that E1047 and E1052Q are critical to TRPM7’s Ca 2+ and Mg 2+ permeability, and its pH sensitivity. Our results indicate that these two glutamate residues are key determinants of both channels’ divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/ pathological functions of TRPM7, and provide molecular insight of the pore architecture of the bi-functional channel kinase TRPM7.