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TRPM7 is an ion channel/protein kinase belonging to TRP melastatin and eEF2 kinase families. Under physiological conditions, most native TRPM7 channels are inactive, due to inhibition by cytoplasmic Mg2+, protons and polyamines. ITRPM7 is strongly potentiated when cell cytosol is depleted of Mg2+ or alkalinized. In Jurkat T cells, Mg2+ inhibition involves a high and a low affinity inhibitor sites, whereas proton inhibition involves only one site. Like many other TRP channels, TRPM7 is activated by PI(4,5)P2 and suppressed by its hydrolysis. Here we examined Mg2+ and pH inhibition of native TRPM7 channels in HEK293 cells overexpressing voltage-sensitive phospholipid phosphatase (VSP) or its catalytically inactive C363S mutant. Phosphoinositide depletion by VSP increased the sensitivity of channels to pH and high Mg2+. Specifically internal pH values that were stimulatory when C363S was expressed (pH 8.2) became inhibitory in the wildtype VSP-expressing cells. 150 uM Mg2+ or pHi 6.5 inhibited ITRPM7 both in wildtype and C363S VSP-expressing cells but with a faster time course in the former group. Both basal and maximum currents were reduced in VSP expressing cells while the mean time to reach maximum amplitude was shortened. In order to prevent the activation of VSP by voltages reaching +85 mV used for recording TRPM7 currents, we tested the effects of VSP on inward currents in divalent cation free solutions by applying voltage ramps reaching only +20 mV. Surprisingly, this command voltage protocol produced results similar to those obtained from ramps reaching +85 mV. These observations suggest that in HEK293 cells, VSP may have significant basal activity even prior to application of depolarizing voltage pulses, possibly due to the depolarized resting membrane potential of these cells. Consistent with this scenario, a subpopulation of wildtype VSP transfected cells behaved like C363S-transfected cells, likely because their resting potential was more negative. Growing HEK cells in 25 mM KCl instead of 5 mM to shift K+ equilibrium potential by ∼+40 mV did not result in increased basal VSP activity, however. In summary, our experiments suggest that voltage-independent Mg2+ and pH inhibition of TRPM7 channels is not direct but, rather, reflects electrostatic screening and disruption of PI(4,5)P2-channel interactions.



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