Abstract
Magnesium is essential for cellular life, but how it is homeostatically controlled still remains poorly understood. Here, we report that members of CNNM family, which have been controversially implicated in both cellular Mg2+ influx and efflux, selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells. Coexpression of CNNMs with the channel markedly increased uptake of divalent cations, which is prevented by an inactivating mutation to the channel's pore. Knockout (KO) of TRPM7 in cells or application of the TRPM7 channel inhibitor NS8593 also interfered with CNNM-stimulated divalent cation uptake. Conversely, KO of CNNM3 and CNNM4 in HEK-293 cells significantly reduced TRPM7-mediated divalent cation entry, without affecting TRPM7 protein expression or its cell surface levels. Furthermore, we found that cellular overexpression of phosphatases of regenerating liver (PRLs), known CNNMs binding partners, stimulated TRPM7-dependent divalent cation entry and that CNNMs were required for this activity. Whole-cell electrophysiological recordings demonstrated that deletion of CNNM3 and CNNM4 from HEK-293 cells interfered with heterologously expressed and native TRPM7 channel function. We conclude that CNNMs employ the TRPM7 channel to mediate divalent cation influx and that CNNMs also possess separate TRPM7-independent Mg2+ efflux activities that contribute to CNNMs' control of cellular Mg2+ homeostasis.
Highlights
More and more it is recognized that cells are molecular machines, with intricate components working seamlessly together to regulate its multitude of activities
Our experiments revealed that overexpression of CNNM2 and CNNM4 with TRPM7 significantly decreased the channel’s protein expression (Fig 1A), which reduced that amount of CNNM2 or CNNM4 that copurified with TRPM7
Using antibodies specific to CNNM3 and CNNM4, 2 isoforms that were identified in the mass spectrometry screening, we found that immunoprecipitation of overexpressed HA-TRPM7 coimmunoprecipitated native CNNM3 and CNNM4 proteins (Fig 1B), indicating that when the channel is overexpressed in HEK-293 cells, it forms complexes with the endogenous CNNM3 and CNNM4 proteins
Summary
More and more it is recognized that cells are molecular machines, with intricate components working seamlessly together to regulate its multitude of activities. Magnesium and other divalent cations are among the cells most essential molecules. With an intracellular concentration of 14 to 20 mM, Mg2+ is the second most abundant cation after K+ [1,2]. Bound to ATP and other macromolecules within the cells, it has been estimated that at least 600 enzymatic reactions are directly or indirectly regulated by Mg2+ [3].
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