Abstract
TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca(2+)-permeable channel activated by oxidative stress or tumor necrosis factoralpha involved in susceptibility to cell death. TRPM2 activation is dependent on the level of intracellular Ca(2+). We explored whether calmodulin (CaM) is the Ca(2+) sensor for TRPM2. HEK 293T cells were transfected with TRPM2 and wild type CaM or mutant CaM (CaM(MUT)) with substitutions of all four EF hands. Treatment of cells expressing TRPM2 with H(2)O(2) or tumor necrosis factor alpha resulted in a significant increase in intracellular calcium ([Ca(2+)](i)). This was not affected by coexpression of CaM, suggesting that endogenous CaM levels are sufficient for maximal response. Cotransfection of CaM(MUT) with TRPM2 dramatically inhibited the increase in [Ca(2+)](i), demonstrating the requirement for CaM in TRPM2 activation. Immunoprecipitation confirmed direct interaction of CaM and CaM(MUT) with TRPM2, and the Ca(2+) 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 H(2)O(2). The increase in [Ca(2+)](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 Ca(2+) 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.
Highlights
Processes including vasoactivation, sensation, fertility, and cell proliferation [1, 3]
TRPM2 is a voltage-independent ion channel known to be regulated by the second messengers ADP-ribose [14, 15] and cyclic adenosine diphosphoribose (cADPR) [21]
Because TRPM2 is involved in the regulation of cell viability following oxidative stress [11, 14, 17], understanding mechanisms involved in TRPM2 activation is of great physiological importance
Summary
Cells Lines, cDNAs, and Transfection Methods—HEK 293T were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum. Western blotting was performed as described above and blots were probed with anti-V5 or anti-CaM antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL. Gel Shift—Three hundred picomoles of purified CaM (Calbiochem) were incubated with different molar ratios of IQ peptide (0–10) in a buffer containing 10 mM Na-HEPES, pH 7.2, and 2 mM CaCl2 or 5 mM EGTA for 30 min at room temperature. Biotinylated Calmodulin Overlay—HEK 293T cells were transfected with V5-TRPM2 or V5-TRPM2-IQMUT1 in pcDNA3.1/V5-His TOPO, lysates were prepared, and immunoprecipitation was performed with anti-V5 antibody. Biotinylated CaM Pull-down with Streptavidin Beads—HEK 293T cells were transfected with V5-TRPM2 or TRPM2-IQMUT1 in pcDNA3.1/ V5-His TOPO. Western blotting was performed with immunoprecipitation pellets, and blots were probed with streptavidin-HRP, or anti-V5-HRP or anti-TRPM2-C followed by the HRP goat anti-rabbit antibody. Statistical significance was determined by unpaired Student’s t test or by one-way analysis of variance
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