TRPM7 silencing attenuates Mg 2+ influx in cardiac myoblasts, H9c2 cells
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(2020) 70:47 Tashiro et al. J Physiol Sci https://doi.org/10.1186/s12576-020-00772-z
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TRPM7 silencing attenuates Mg2+ influx in cardiac myoblasts, H9c2 cells Michiko Tashiro1* , Masato Konishi1, Ryo Kobayashi2, Hana Inoue1 and Utako Yokoyama1
Abstract TRPM7, a member of the melastatin subfamily of transient receptor potential channels, is suggested to be a potential candidate for a physiological Mg2+ channel. However, there is no direct evidence of Mg2+ permeation through endogenous TRPM7. To determine the physiological roles of TRPM7 in intracellular Mg2+ homeostasis, we measured the cytoplasmic free Mg2+ concentration ( [Mg2+]i) in TRPM7-silenced H9c2 cells. [Mg2+]i was measured in a cluster of 8–10 cells using the fluorescent indicator, furaptra. TRPM7 silencing did not change [ Mg2+]i in Ca2+-free Tyrode’s solution containing 1 mM M g2+. Increasing the extracellular Mg2+ to 92.5 mM raised [Mg2+]i in control cells (1.56 ± 0.19 mM) at 30 min, while this effect was significantly attenuated in TRPM7-silenced cells (1.12 ± 0.07 mM). The Mg2+ efflux driven by Na+ gradient was unaffected by TRPM7 silencing. These results suggest that TRPM7 regulates the rate of M g2+ influx in H9c2 cells, although cytoplasmic Mg2+ homeostasis at basal conditions is unaffected by TRPM7 silencing. Keywords: Magnesium, TRPM7, Cardiac myoblast, H9c2, Mag-fura-2 Background The importance of intracellular Mg2+ has been widely recognized. Mg2+ is essential for protein synthesis, the regulation of ion channels, and as a co-factor in over 600 enzymatic reactions, many of which affect cellular functions and viability [1]. Thus, cytoplasmic free Mg2+ concentration ([Mg2+]i) should be kept in physiological range, but the molecules responsible for Mg2+ influx pathway remain to be identified. In cardiac myocytes, several candidates, such as transient receptor potential melastatin subfamily member 7 (TRPM7, non-selective cation channel) and magnesium transporter 1 (MagT1, Mg2+-selective channel), have been proposed [2, 3]. The properties of these channels have been investigated by heterologous overexpression systems [4, 5], and the data driven by these types of studies may not always be
associated with the physiological functions of endogenous Mg2+-related channels. The low rate of Mg2+ influx under physiological conditions makes it difficult to identify endogenous Mg2+ channels. We previously quantified M g2+ entry in rat ventricular myocytes, and found that the rate of M g2+ influx was altered by TRPM7 modulators in a concentration-dependent manner with EC50 (half-maximal effective concentration) values comparable with those reported for the TRPM7 channel activities [6, 7]. To extend these pharmacological studies suggesting that TRPM7 functions as a physiological pathway of Mg2+ influx in native cardiac myocytes, we investigated the rate of Mg2+ transport of the cardiac myoblasts (H9c2) transfected with TRPM7-targeted shRNA. Some of the results have been reported in abstract f
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