Four Ca2+ Ions Activate TRPM2 channels by binding in deep crevices near the pore but intracellularly of the gate

Abstract

TRPM2 is a tetrameric Ca2+ -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 Ca2+ , but the mechanism and the binding sites for Ca2+ 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 Ca2+ activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca2+ activated ̃ 50-pS nonselective cation channels; K 1/2 for ADPR was ̃1 μ M at saturating Ca2+ . Intracellular Ca2+ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca2+ ) reveals that Ca2+ activation is a consequence of tighter binding of Ca2+ in the open rather than in the closed channel conformation. Four Ca2+ 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 Ca2+ on the extracellular side clearly show that closed channels do not sense extracellular Ca2+ , but once channels have opened Ca2+ entering passively through the pore slows channel closure by keeping the " activating sites " saturated, despite rapid continuous Ca2+ -free wash of the intracellular channel surface. This effect of extracellular Ca2+ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca2+ infl ux 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 Ca2+ likely triggers prolonged, self-sustained TRPM2 activity.© 2009 Csanády and Töröcsik.