Transient receptor potential melastatin 2 (TRPM2) is a Ca
2+-permeable cation channel expressed in immune cells of phagocytic lineage, pancreatic β cells, and brain neurons and is activated under oxidative stress. TRPM2 activity is required for immune cell activation and insulin secretion and is responsible for postischemic neuronal cell death. TRPM2 is opened by binding of ADP ribose (ADPR) to its C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain, which, when expressed in isolation, cleaves ADPR into AMP and ribose-5-phosphate. A suggested coupling of this enzymatic activity to channel gating implied a potentially irreversible gating cycle, which is a unique feature of a small group of channel enzymes known to date. The significance of such a coupling lies in the conceptually distinct pharmacologic strategies for modulating the open probability of channels obeying equilibrium versus nonequilibrium gating mechanisms. Here we examine the potential coupling of TRPM2 enzymatic activity to pore gating. Mutation of several residues proposed to enhance or eliminate NUDT9-H catalytic activity all failed to affect channel gating kinetics. An ADPR analog, α-β-methylene-ADPR (AMPCPR), was shown to be entirely resistant to hydrolysis by NUDT9, but nevertheless supported TRPM2 channel gating, albeit with reduced apparent affinity. The rate of channel deactivation was not slowed but, rather, accelerated in AMPCPR. These findings, as well as detailed analyses of steady-state gating kinetics of single channels recorded in the presence of a range of concentrations of ADPR or AMPCPR, identify TRPM2 as a simple ligand-gated channel that obeys an equilibrium gating mechanism uncoupled from its enzymatic activity.Transient receptor potential melastatin 2 (TRPM2) belongs to the TRP protein family and is abundantly expressed in brain neurons, bone marrow, phagocytes, pancreatic β cells, and cardiomyocytes, where it forms Ca
2+-permeable nonselective cation channels that open under oxidative stress. On contact with pathogens, phagocytic cells produce reactive oxygen species (ROS); the resulting activation of TRPM2 provides the Ca
2+ influx necessary for cell migration and chemokine production (
1). In pancreatic β cells, TRPM2 activity contributes to glucose-evoked insulin secretion; TRPM2 knock-out mice show higher resting blood glucose levels and impaired glucose tolerance (
2).TRPM2 activity is also linked to several pathologic conditions that lead to apoptosis (
3). Reperfusion after ischemia results in ROS generation; consequent Ca
2+ influx through TRPM2 causes Ca
2+ dysregulation and cell death. Certain neurodegenerative diseases, such as Alzheimer’s disease, also involve oxidative stress and TRPM2 activation. In contrast, a loss-of-function TRPM2 mutation identified in patients with amyotrophic lateral sclerosis and Parkinson''s disease dementia (
4), as well as two TRPM2 mutations associated with bipolar disorder (
5), suggest loss of TRPM2 activity can also cause disease.Similar to most TRP family ion channels, the TRPM2 channel is a homotetramer, and its transmembrane (TM) architecture resembles that of voltage-gated cation channels (
6,
7). In addition to the TM domain and an N-terminal cytosolic domain of unknown function, TRPM2 contains an ∼270-residue C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain. 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). 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. This “T5L” TRPM2 variant, which shows no rundown but preserves intact regulation of gating by Ca
2+ and ADPR (
11), provides an unprecedented opportunity to study TRPM2 gating at steady state.Early studies reported slow (∼0.1 s
−1) but detectable ADPRase activity of isolated purified NUDT9-H (
8,
12), classifying TRPM2 into the special group of channel-enzymes (“chanzymes”) that includes TRPM6 and TRPM7 (
3) and the CFTR cystic fibrosis transmembrane conductance regulator (CFTR) chloride ion channel (
13). TRPM2 pore opening/closure happens on the timescale of the reported ADPRase activity (
11), which is consistent with coupling between gating and catalytic activity, as demonstrated for CFTR in which pore gating follows an irreversible cycle tightly linked to ATP binding and hydrolysis at conserved cytosolic domains (
14).The involvement of TRPM2 in multiple diseases has made it an emerging therapeutic target. Depending on the disease, both inhibition (e.g., stroke, myocardial infarction, Alzheimer’s disease, chronic inflammation, hyperinsulinism) and stimulation (e.g., diabetes, amyotrophic lateral sclerosis, Parkinson''s disease dementia, bipolar disorder) of TRPM2 activity might be useful therapeutically. Because TRP family channels are involved in diverse processes (
3), any useful TRPM2 agonists/antagonists will need to be highly selective. This singles out the NUDT9-H domain, the component unique to TRPM2, as the most attractive drug target. The significance of understanding whether ADPRase activity and gating are coupled is that optimal strategies for modulating fractional occupancy of a particular conformational state are profoundly different for equilibrium systems than for nonequilibrium systems. For most ion channels, pore gating is an equilibrium process, and open probability is modulated simply by energetic stabilization of either open (activators) or closed (inhibitors) channel ground states. In contrast, channels that gate by a nonequilibrium cycle are most efficiently accumulated in either open or closed states by manipulating the stability of transition states for rate-limiting irreversible steps (
15). The aim of this study was to examine the tightness of coupling between the ADPRase cycle and specific gating transitions in TRPM2.
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