Extended Data Fig. 8: Electrophysiological experiments.

a, b, Alanine mutations were introduced within the ADPR-binding pocket and showed expression levels comparable to that of wild-type TRPM2, observed from both transfected cells (a) and fluorescence-detection size-exclusion chromatography (b). c, d, Electrophysiological experiments were carried out to measure the amplitude of agonist-induced current in inside-out patches pulled from HEK293 cells, with c showing the representative current and d showing the statistics of current amplitude and cell numbers. At +60 mV, robust current (4.87 ± 0.55 nA, n = 9 cells) could be detected when applying 0.1 mM ADPR and 1 mM Ca²⁺ onto inside-out patches expressing wild-type TRPM2. Single mutations (K154A, Y271A, E274A, R278A and R334A) each show robust channel activation (n = 5 cells, 8 cells, 8 cells, 7 cells and 8 cells for the corresponding mutants) and—other than K154A receptors, which did not show a markedly reduced current amplitude—mean current amplitudes for the mutated receptors were around two- to threefold smaller compared to the wild type. Introducing double mutations R278A/R334A (n = 7 cells) to the receptor nearly abolished ADPR/Ca²⁺-induced current. Deletion of the NUDT9-H domain (∆NUDT9-H) (n = 6 cells) also nearly abolished the ADPR/Ca²⁺-induced current. Data are shown as mean ± s.e.m.