Alanine substitution scanning of 11 identified residues in ADPR-binding pockets. (A) 100 µM ADPR-induced TRPM2 currents traces at −80 mV (left panel) and the I-V relationship curves (right panel, at time points indicated by a1 pointed to before pH 5.0 treatment and b1 pointed to after pH 5.0 inhibiting TRPM2 channel). W.C., whole cell. (B) The concentration–current response relationship for ADPR and the WT TRPM2 channel. Data are expressed as mean ± SEM from seven independent repetitions. (C) Summary of the currents induced by EC₁₀ (gray) and EC₉₀ (black) concentrations of ADPR. Data are expressed as mean ± SEM from at least five independent repetitions. (D) Biotinylation assay for surface expression of the TRPM2 and its mutants. The eight mutants that exhibited no channel function were expressed on the cell surface in HEK293 cells. The top panel shows surface expression, and the bottom panel shows total expression. Arrows indicate the specific band size of WT and the indicated TRPM2 mutants.