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László Csanády and Beáta Törcsik

Department of Medical Biochemistry, Semmelweis University, Budapest, H-1094, Hungary

Correspondence to László Csanády: laszlo.csanady{at}eok.sote.hu

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 Ca²⁺ 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 µM 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 10⁶-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⁺ 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 Ca²⁺ likely triggers prolonged, self-sustained TRPM2 activity.

© 2009 Csanády and Törcsik
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