Physiological Pathway of Magnesium Influx in Rat Ventricular Myocytes

Abstract

Cytoplasmic free Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with a fluorescent indicator furaptra (mag-fura-2) introduced by AM-loading. By incubation of the cells in a high-K+ (Ca2+- and Mg2+-free) solution, [Mg2+]i decreased from ∼ 0.9 mM to 0.2 to 0.5 mM. The lowered [Mg2+]i was 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 being proportional to the initial Mg2+ influx rate. The Mg2+ influx rate was inversely related to [Mg2+]i, being higher at low [Mg2+]i. The Mg2+ influx rate was augmented by the high extracellular Mg2+ concentration (5 mM), whereas it was greatly reduced by cell membrane depolarization caused by high K+. Known inhibitors of TRPM7 channels, 2-aminoethoxydiphenyl borate (2-APB), NS8593, and spermine reduced the Mg2+ influx rate with half inhibitory concentrations (IC50) of, respectively, 17 μM, 2.0 μM, and 22 μM. We also studied Ni2+ influx by fluorescence quenching of intracellular furaptra by Ni2+. The Ni2+ influx was activated by lowering intra- and extracellular Mg2+ concentrations, and it was inhibited by 2-APB and NS8593 with IC50 values comparable with those for the Mg2+ influx. Intracellular alkalization (caused by pulse application of NH4Cl) enhanced, whereas intracellular acidification (induced after the removal of NH4Cl) slowed the Mg2+ influx. Under the whole-cell patch-clamp configuration, the removal of intracellular and extracellular divalent cations induced large inward and outward currents, MIC (Mg-inhibited cation) currents or IMIC, carried by monovalent cations likely via TRPM7 channels. IMIC measured at -120 mV was diminished to ∼ 50% by 100 μM 2-APB or 10 μM NS8593. These results suggest that TRPM7/MIC channels serve as a major physiological pathway of Mg2+ influx in rat ventricular myocytes.