Tashiro Michiko, Inoue Hana, Konishi Masato
Department of Physiology, Tokyo Medical University, Tokyo 160-8402, Japan.
Department of Physiology, Tokyo Medical University, Tokyo 160-8402, Japan.
Biophys J. 2014 Nov 4;107(9):2049-58. doi: 10.1016/j.bpj.2014.09.015.
Cytoplasmic free Mg(2+) concentration ([Mg(2+)]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(+) (Ca(2+)- and Mg(2+)-free) solution, [Mg(2+)]i decreased from ? 0.9 mM to 0.2 to 0.5 mM. The lowered [Mg(2+)]i was recovered by perfusion with Ca(2+)-free Tyrode's solution containing 1 mM Mg(2+). The time course of the [Mg(2+)]i recovery was fitted by a single exponential function, and the first derivative at time 0 was analyzed as being proportional to the initial Mg(2+) influx rate. The Mg(2+) influx rate was inversely related to [Mg(2+)]i, being higher at low [Mg(2+)]i. The Mg(2+) influx rate was augmented by the high extracellular Mg(2+) 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 Mg(2+) influx rate with half inhibitory concentrations (IC50) of, respectively, 17 ?M, 2.0 ?M, and 22 ?M. We also studied Ni(2+) influx by fluorescence quenching of intracellular furaptra by Ni(2+). The Ni(2+) influx was activated by lowering intra- and extracellular Mg(2+) concentrations, and it was inhibited by 2-APB and NS8593 with IC50 values comparable with those for the Mg(2+) influx. Intracellular alkalization (caused by pulse application of NH4Cl) enhanced, whereas intracellular acidification (induced after the removal of NH4Cl) slowed the Mg(2+) 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 ?M 2-APB or 10 ?M NS8593. These results suggest that TRPM7/MIC channels serve as a major physiological pathway of Mg(2+) influx in rat ventricular myocytes.
采用AM负载法将荧光指示剂呋喃普特拉（镁-双波长比率荧光指示剂-2）导入大鼠心室肌细胞，测定细胞内游离镁离子浓度（[Mg(2+)]i）。将细胞置于高钾（无钙和镁）溶液中孵育，[Mg(2+)]i从约0.9 mM降至0.2至0.5 mM。通过用含1 mM镁的无钙台氏液灌注，可使降低的[Mg(2+)]i恢复。[Mg(2+)]i恢复的时间进程可用单指数函数拟合，并将时间0时的一阶导数分析为与初始镁离子内流速率成正比。镁离子内流速率与[Mg(2+)]i呈负相关，在低[Mg(2+)]i时较高。高细胞外镁离子浓度（5 mM）可增加镁离子内流速率，而高钾引起的细胞膜去极化则使其大幅降低。已知的瞬时受体电位M型7通道（TRPM7）抑制剂2-氨基乙氧基二苯硼酸（2-APB）、NS8593和精胺可降低镁离子内流速率，其半数抑制浓度（IC50）分别为17 μM、2.0 μM和22 μM。我们还通过镍离子对细胞内呋喃普特拉的荧光猝灭研究了镍离子内流。降低细胞内和细胞外镁离子浓度可激活镍离子内流，2-APB和NS8593可抑制镍离子内流，其IC50值与镁离子内流的IC50值相当。细胞内碱化（由氯化铵脉冲施加引起）增强镁离子内流，而细胞内酸化（氯化铵去除后诱导）则减缓镁离子内流。在全细胞膜片钳配置下，去除细胞内和细胞外二价阳离子会诱导大的内向和外向电流，即镁离子抑制阳离子（MIC）电流或IMIC，可能由单价阳离子通过TRPM7通道携带。在-120 mV下测量的IMIC，100 μM 2-APB或10 μM NS8593可使其降低至约50%。这些结果表明，TRPM7/MIC通道是大鼠心室肌细胞镁离子内流的主要生理途径。
