Effects of TRPM7 Inhibitors on Physiological Mg2+ Influx in Rat Ventricular Myocytes

Abstract

We measured free Mg2+ concentration ([Mg2+]i) in rat ventricular myocytes using a fluorescent indicator furaptra (mag-fura-2). [Mg2+]i decreased from ∼0.9 mM to 0.2-0.5 mM by incubation of the cells in a high-K+ (Ca2+- and Mg2+-free) solution, and recovered by perfusion with Ca2+-free Tyrode's solution containing 1 mM Mg2+. The time course of the [Mg2+]i recovery was fitted by a single exponential function, and the first derivative at time 0 was analyzed as an initial Mg2+ influx rate. In order to characterize physiological Mg2+ influx pathways, we used known TRPM7 inhibitors, 2-Aminoethoxydiphenyl borate (2-APB) and NS8593. The initial rate of Mg2+ influx was decreased to 43±10 % (n=6) by 100 μM 2-APB, and to 12±8.6 % (n=5) by 10 μM NS8593. These compounds inhibited the Mg2+ influx with half inhibitory concentrations (IC50) of 17 μM (2-APB) and 2.0 μM (NS8593). 2-APB and NS8593 also inhibited Ni2+ influx when estimated by quenching of furaptra fluorescence with IC50 values of, respectively, 20 μM and 4.4 μM; these values are comparable to those for Mg2+ influx. Under the whole-cell patch-clamp configuration, removal of intracellular and extracellular divalent cations induced large inward and outward currents, IMIC, carried by monovalent cations likely via TRPM7 channels. The IMIC measured at −120 mV was diminished to 48±3.6 % (n=7) by 100 μM 2-APB, and to 50±12 % (n=4) by 10 μM NS8593. These results support our previous conclusion [Biophys J 102:664a, 2012] that TRPM7/MIC channels serve as a major physiological pathway of Mg2+ influx in rat ventricular myocytes.