Lys¹¹¹⁰ of TRPM2 is critical for channel activation

TRPM2 (transient receptor potential melastatin 2) is a nonselective Ca ²⁺ -permeable cation channel activated by ADPR (adenosine diphosphoribose) and H₂O₂. It is widely expressed in mammalian cells and plays an important role in the regulation of various cell functions. However, the mechanisms of TRPM2 channel activation are not fully understood. Previously, we reported that TRPM2 channel activation is induced by high intracellular Cl- concentration. In the present study, we investigated the functional role of Lys¹¹¹⁰ in the membraneproximal C-terminal region by site-directed mutagenesis. Replacement of the positively charged amino acid lysine (Lys¹¹¹⁰) with the neutrally charged amino acid asparagine (K1110N) or the negatively charged amino acid glutamic acid (K1110E) generated mutants that failed to induce an increase in free cytosolic calcium concentration ([Ca²⁺ ]_i) not only by intracellular injection of Cl^- , but also by H₂O₂ or ADPR. However, a mutant generated by replacing the lysine residue with a positively charged amino acid arginine (K1110R) displayed channel activity similar to wild-type TRPM2. Interestingly, in the K1107N/K1110N doublepoint mutant, the impaired function of the K1110N mutant in response to ADPR andH ₂O₂, but not to Cl^- ,was recovered. There were no changes in protein expression, membrane trafficking and oligomerization of the mutant channels. The extent of [Ca²⁺ ]_i increase by H₂O₂ in HEK (human embryonic kidney)-293 cells expressing TRPM2 mutants was well correlated with the degree of susceptibility to H ₂O₂-induced cell death. These results display the crucial role of a positively charged amino acid residue at position 1110 for TRPM2 channel activity.