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 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; K1/2 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.

Putative chanzyme activity of TRPM2 cation channel is unrelated to pore gating

“…6G). In contrast, coactivating Ca 2+ ions were shown to remain at rapid equilibrium with the bulk cytosolic solution, even while a TRPM2 channel is open (10).…”

“…The latter shows high (∼50%) sequence homology to the soluble mitochondrial enzyme NUDT9, an active ADP ribose (ADPR) pyrophosphatase (ADPRase) from the Nudix hydrolase family, which splits ADPR into AMP and ribose-5-phosphate (8). TRPM2 channels are coactivated by ADPR binding to NUDT9-H (9) and by Ca 2+ binding to unidentified intracellular binding sites (10). ADPR is the key that links TRPM2 activation to oxidative stress; in living cells exposed to ROS, ADPR is released from mitochondria (9).…”

“…ADPR is the key that links TRPM2 activation to oxidative stress; in living cells exposed to ROS, ADPR is released from mitochondria (9). In the past, studying TRPM2 channel gating at steady state has been limited by rapid deactivation of TRPM2 currents in cell-free patches (10). This rundown was recently shown to involve a conformational change of the ion selectivity filter, which could be completely prevented by a pore-loop substitution.…”

Identification of Direct and Indirect Effectors of the Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel

“…The primary activators of TRPM2 are ADP-ribose (ADPR) and Ca 2ϩ (2)(3)(4); simultaneous binding of both agonists is required for channel opening (9). TRPM2 channels are homotetramers (10), and ADPR binds to the NUDT9-H domains located at the cytosolic C termini of the subunits, named based on sequence homology to the mitochondrial enzyme NUDT9 (2).…”

A TRP channel contributes to insulin secretion by pancreatic β-cells

“…[10][11][12][13] More recently, it has been reported that TRPM2 is activated by intracellular Ca 2+ . 14,15 TRPM4 is widely distributed and has some of the features of the Ca-NS of pancreatic cells, such as block by adenine nucleotides. 16 However, animals that carry a targeted deletion of TRPM4 do not have any defects in glucose tolerance or insulin secretion.…”