Balázs Tóth

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

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca2+ and ADP-ribose (ADPR) but also modulated in intact cells by several additional factors. Superfusion of TRPM2-expressing cells with H2O2 or intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) activates, whereas dialysis of AMP inhibits, TRPM2 whole-cell currents. Additionally, H2O2, cADPR, and NAADP enhance ADPR sensitivity of TRPM2 currents in intact cells. Because in whole-cell recordings the entire cellular machinery for nucleotide and Ca2+ homeostasis is intact, modulators might affect TRPM2 activity either directly, by binding to TRPM2, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca2+. To identify direct modulators of TRPM2, we have studied the effects of H2O2, AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide in inside-out patches from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H2O2 (1 mm) and enzymatically purified cADPR (10 μm) failed to activate, whereas AMP (200 μm) failed to inhibit TRPM2 currents. NAADP was a partial agonist (maximal efficacy, ∼50%), and nicotinic acid adenine dinucleotide was a full agonist, but both had very low affinities (K0.5 = 104 and 35 μm). H2O2, cADPR, and NAADP did not enhance activation by ADPR. Considering intracellular concentrations of these compounds, none of them are likely to directly affect the TRPM2 channel protein in a physiological context.

Pore collapse underlies irreversible inactivation of TRPM2 cation channel currents

The Ca2+-permeable cation channel transient receptor potential melastatin 2 (TRPM2) plays a key role in pathogen-evoked phagocyte activation, postischemic neuronal apoptosis, and glucose-evoked insulin secretion, by linking these cellular responses to oxidative stress. TRPM2 channels are coactivated by binding of intracellular ADP ribose and Ca2+ to distinct cytosolically accessible sites on the channels. These ligands likely regulate the activation gate, conserved in the voltage-gated cation channel superfamily, that comprises a helix bundle formed by the intracellular ends of transmembrane helix six of each subunit. For several K+ and TRPM family channels, activation gate opening requires the presence of phosphatidylinositol-bisphosphate (PIP2) in the inner membrane leaflet. Most TRPM family channels inactivate upon prolonged stimulation in inside-out patches; this “rundown” is due to PIP2 depletion. TRPM2 currents also run down within minutes, but the molecular mechanism of this process is unknown. Here we report that high-affinity PIP2 binding regulates Ca2+ sensitivity of TRPM2 activation. Nevertheless, TRPM2 inactivation is not due to PIP2 depletion; rather, it is state dependent, sensitive to permeating ions, and can be completely prevented by mutations in the extracellular selectivity filter. Introduction of two negative charges plus a single-residue insertion, to mimic the filter sequence of TRPM5, results in TRPM2 channels that maintain unabated maximal activity for over 1 h, and display altered permeation properties but intact ADP ribose/Ca2+-dependent gating. Thus, upon prolonged stimulation, the TRPM2 selectivity filter undergoes a conformational change reminiscent of that accompanying C-type inactivation of voltage-gated K+ channels. The noninactivating TRPM2 variant will be invaluable for gating studies.

Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2′-phosphate

ADP-ribose-2′-phosphate acts as a direct agonist of TRPM2, whereas NAD, NAAD, and NAADP do not.

Ca2+ release triggered by NAADP in hepatocyte microsomes.

NAADP (nicotinic acid-adenine dinucleotide phosphate) is fast emerging as a new intracellular Ca2+-mobilizing messenger. NAADP induces Ca2+ release by a mechanism that is distinct from IP3 (inositol 1,4,5-trisphosphate)- and cADPR (cADP-ribose)-induced Ca2+ release. In the present study, we demonstrated that micromolar concentrations of NAADP trigger Ca2+ release from rat hepatocyte microsomes. Cross-desensitization to IP3 and cADPR by NAADP did not occur in liver microsomes. We report that non-activating concentrations of NAADP can fully inactivate the NAADP-sensitive Ca2+-release mechanism in hepatocyte microsomes. The ability of thapsigargin to block the NAADP-sensitive Ca2+ release is not observed in sea-urchin eggs or in intact mammalian cells. In contrast with the Ca2+ release induced by IP3 and cADPR, the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ concentration and pH (in the range 6.4-7.8). The NAADP-elicited Ca2+ release cannot be blocked by the inhibitors of the IP3 receptors and the ryanodine receptor. On the other hand, verapamil and diltiazem do inhibit the NAADP- (but not IP3- or cADPR-) induced Ca2+ release.

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

Significance Ion channels are protein pores that allow passive transmembrane ion flow. These pores are opened and closed (gated) by various cellular signals. Typically, the mechanism of gating conformational changes is an equilibrium process, but for some channels, gating is an irreversible cycle, for example, linked to an enzymatic activity. For equilibrium mechanisms, channel activity is readily modulated by energetic stabilization of closed or open states, whereas for cyclic gating, alteration of transition-state stabilities most effectively modulates activity. Transient receptor potential melastatin 2 (TRPM2), a cation channel involved in multiple physiologic and pathophysiologic processes, possesses enzymatic activity, which cleaves its activating ligand. This work addresses, and rules out, a suggested link between that catalysis and pore gating in TRPM2, classifying it among the channels that gate at equilibrium. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-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.

Direct and Indirect Effectors of the TRPM2 Cation Channel

TRPM2 is a Ca2+ permeable cation channel which plays a role in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca2+ and ADP ribose (ADPR). In addition, in intact cells, a large number of compounds appear to modulate TRPM2 activity. Superfusion of TRPM2-expressing cells with hydrogen-peroxide (H2O2) activates TRPM2 currents, just as intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP). Importantly, H2O2, cADPR, and NAADP enhance ADPR-induced TRPM2 whole-cell currents. Finally, in intact cells AMP acts as a TRPM2 inhibitor. Because in whole-cell recordings the entire cellular machinary involved in nucleotide- and Ca2+-homeostasis is in place, compounds might affect TRPM2 activity either directly, by binding to the TRPM2 protein, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca2+. To identify direct modulators of TRPM2 activity, we have studied the effects of H2O2, AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide (NAAD) in inside-out patches excised from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H2O2 (1 mM) and enzymatically purified cADPR (10 μM) failed to activate, while AMP (200 μM) failed to inhibit TRPM2 currents. NAADP acted as a partial agonist (maximal efficacy ∼50%) while NAAD was a full agonist, but both with low affinities (K0.5=104 and 35 μM). Neither of H2O2, cADPR, and NAADP enhanced activation by ADPR. Thus, in a physiological context the above compounds do not directly affect the TRPM2 channel protein. [OTKA grant F68143]