Depletion of plasma membrane-associated phosphoinositides mimics inhibition of TRPM7 channels by cytosolic Mg2+, spermine, and pH

Abstract

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is an ion channel/protein kinase belonging to the TRP melastatin and eEF2 kinase families. Under physiological conditions, most native TRPM7 channels are inhibited by cytoplasmic Mg²⁺, protons, and polyamines. Currents through these channels (I_TRPM7) are robustly potentiated when the cell interior is exchanged with low Mg²⁺-containing buffers. I_TRPM7 is also potentiated by phosphatidyl inositol bisphosphate (PI(4,5)P₂) and suppressed by its hydrolysis. Here we characterized internal Mg²⁺- and pH-mediated inhibition of TRPM7 channels in HEK293 cells overexpressing WT voltage-sensing phospholipid phosphatase (VSP) or its catalytically inactive variant VSP-C363S. VSP-mediated depletion of membrane phosphoinositides significantly increased channel sensitivity to Mg²⁺ and pH. Proton concentrations that were too low to inhibit I_TRPM7 when the VSP-C363S variant was expressed (pH 8.2) became inhibitory in WT VSP-expressing cells. At pH 6.5, protons inhibited I_TRPM7 both in WT and VSP C363S-expressing cells but with a faster time course in the WT VSP-expressing cells. Inhibition by 150 μm Mg²⁺ was also significantly faster in the WT VSP-expressing cells. Cellular PI(4,5)P₂ depletion increased the sensitivity of TRPM7 channels to the inhibitor 2-aminoethyl diphenyl borinate, which acidifies the cytosol. Single substitutions at Ser-1107 of TRPM7, reducing its sensitivity to Mg²⁺, also decreased its inhibition by spermine and acidic pH. Furthermore, these channel variants were markedly less sensitive to VSP-mediated PI(4,5)P₂ depletion than the WT. We conclude that the internal Mg²⁺-, polyamine-, and pH-mediated inhibition of TRPM7 channels is not direct but, rather, reflects electrostatic screening and resultant disruption of PI(4,5)P₂-channel interactions.

Keywords: TRPM; channel activation; gain-of-function mutation; magnesium; mutant; phosphoinositide; polyamine; transfection; transient receptor potential channels (TRP channels); voltage-sensitive phosphatase.