Four Ca ²⁺ Ions Activate TRPM2 Channels by Binding in Deep Crevices near the Pore but Intracellularly of the Gate

Abstract

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 ∼50-pS nonselective cation channels; K _1/2 for ADPR was ∼1 µM 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 ∼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.