Regulation of the transient receptor potential channel TRPM2 by the Ca2+ sensor calmodulin

TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca2+-permeable channel activated by oxidative stress or tumor necrosis factor alpha involved in susceptibility to cell death. TRPM2 activation is dependent on the level of intracellular Ca2+. We explored whether calmodulin (CaM) is the Ca2+ sensor for TRPM2. HEK 293T cells were transfected with TRPM2 and wild type CaM or mutant CaM (CaMMUT) with substitutions of all four EF hands. Treatment of cells expressing TRPM2 with H2O2 or tumor necrosis factor alpha resulted in a significant increase in intracellular calcium ([ Ca2+](i)). This was not affected by coexpression of CaM, suggesting that endogenous CaM levels are sufficient for maximal response. Cotransfection of CaMMUT with TRPM2 dramatically inhibited the increase in [Ca2+](i), demonstrating the requirement for CaM in TRPM2 activation. Immunoprecipitation confirmed direct interaction of CaM and CaMMUT with TRPM2, and the Ca2+ dependence of this association. CaM bound strongly to the TRPM2 N terminus (amino acids 1-730), but weakly to the C terminus (amino acids 1060-1503). CaM binding to an IQ-like motif (amino acids 406-416) in the TRPM2 N terminus was demonstrated utilizing gel shift, immunoprecipitation, biotinylated CaM overlay, and pull-down assays. A substitution mutant of the IQ-like motif of TRPM2 (TRPM2-IQ(MUT1)) reduced but did not eliminate CaM binding to TRPM2, suggesting the presence of at least one other CaM binding site. The functional importance of the TRPM2 IQ-like motif was demonstrated by treatment of TRPM2-IQ(MUT1)-expressing cells with H2O2. The increase in [Ca2+](i) observed with wild type TRPM2 was absent and cell viability was preserved. These data demonstrate the requirement for CaM in TRPM2 activation. They suggest that Ca2+ entering through TRPM2 enhances interaction of CaM with TRPM2 at the IQ-like motif in the N terminus, providing crucial positive feedback for channel activation.