TRPM2 is a tetrameric Ca(2+)-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca(2+), but the mechanism and the binding sites for Ca(2+) activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca(2+) dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca(2+) activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca(2+) activated approximately 50-pS nonselective cation channels; K(1/2) for ADPR was approximately 1 microM at saturating Ca(2+). Intracellular Ca(2+) dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca(2+)) reveals that Ca(2+) activation is a consequence of tighter binding of Ca(2+) in the open rather than in the closed channel conformation. Four Ca(2+) ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant approximately 33-fold, producing altogether 10(6)-fold activation. Experiments in the presence of 1 mM of free Ca(2+) on the extracellular side clearly show that closed channels do not sense extracellular Ca(2+), but once channels have opened Ca(2+) entering passively through the pore slows channel closure by keeping the "activating sites" saturated, despite rapid continuous Ca(2+)-free wash of the intracellular channel surface. This effect of extracellular Ca(2+) on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca(2+) influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single brief puff of Ca(2+) likely triggers prolonged, self-sustained TRPM2 activity.