title: Four Ca2+ 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 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.
authors.affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary. laszlo.csanady@eok.sote.hu
authors.forename: László
authors.initials: L
authors.lastname: Csanády
authors.forename: Beáta
authors.initials: B
authors.lastname: Törocsik
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D025461:Feedback, Physiological / Q000502:physiology; D005260:Female; D006801:Humans; D015640:Ion Channel Gating; D008564:Membrane Potentials; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D013329:Structure-Activity Relationship; D050053:TRPM Cation Channels / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D013816:Thermodynamics; D014982:Xenopus laevis
chemical_list: D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D000246:Adenosine Diphosphate Ribose; D002118:Calcium