Structure of a TRPM2 channel in complex with Ca2+ explains unique gating regulation.
ABSTRACT: Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 is activated by cytosolic Ca2+, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here, we present the ~3 Å resolution electron cryo-microscopic structure of TRPM2 from Nematostella vectensis, 63% similar in sequence to human TRPM2, in the Ca2+-bound closed state. Compared to other TRPM channels, TRPM2 exhibits unique structural features that correlate with its function. The pore is larger and more negatively charged, consistent with its high Ca2+ selectivity and larger conductance. The intracellular Ca2+ binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca2+. In addition, the absence of a post-filter motif is likely the cause of the rapid inactivation of human TRPM2. Together, our cryo-EM and electrophysiology studies provide a molecular understanding of the unique gating mechanism of TRPM2.
Project description:Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated with numerous diseases. It has a C-terminal NUDT9 homology (NUDT9H) domain responsible for binding adenosine diphosphate (ADP)-ribose (ADPR), and both ADPR and calcium (Ca2+) are required for TRPM2 activation. Here we report cryo-electron microscopy structures of human TRPM2 alone, with ADPR, and with ADPR and Ca2+ NUDT9H forms both intra- and intersubunit interactions with the N-terminal TRPM homology region (MHR1/2/3) in the apo state but undergoes conformational changes upon ADPR binding, resulting in rotation of MHR1/2 and disruption of the intersubunit interaction. The binding of Ca2+ further engages transmembrane helices and the conserved TRP helix to cause conformational changes at the MHR arm and the lower gating pore to potentiate channel opening. These findings explain the molecular mechanism of concerted TRPM2 gating by ADPR and Ca2+ and provide insights into the gating mechanism of other TRP channels.
Project description:Transient Receptor Potential Melastatin 2 (TRPM2) is a cation channel important for the immune response, insulin secretion, and body temperature regulation. It is activated by cytosolic ADP ribose (ADPR) and contains a nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain homologous to ADPR phosphohydrolases (ADPRases). Human TRPM2 (hsTRPM2) is catalytically inactive due to mutations in the conserved Nudix box sequence. Here, we show that TRPM2 Nudix motifs are canonical in all invertebrates but vestigial in vertebrates. Correspondingly, TRPM2 of the cnidarian Nematostella vectensis (nvTRPM2) and the choanoflagellate Salpingoeca rosetta (srTRPM2) are active ADPRases. Disruption of ADPRase activity fails to affect nvTRPM2 channel currents, reporting a catalytic cycle uncoupled from gating. Furthermore, pore sequence substitutions responsible for inactivation of hsTRPM2 also appeared in vertebrates. Correspondingly, zebrafish (Danio rerio) TRPM2 (drTRPM2) and hsTRPM2 channels inactivate, but srTRPM2 and nvTRPM2 currents are stable. Thus, catalysis and pore stability were lost simultaneously in vertebrate TRPM2 channels.
Project description:The archetypal TRPM2-like channel of the sea anemone Nematostella vectensis is gated by ADPR like its human orthologue but additionally exhibits properties of other vertebrate TRPM channels. Thus it can help towards an understanding of gating and regulation of the whole subfamily. To elucidate further the role of Ca2+ as a co-factor of ADPR, we exploited 2-aminoethyl diphenylborinate (2-APB), previously shown to exert either inhibitory or stimulatory effects on diverse TRPM channels, or both in a concentration-dependent manner. 2-APB in high concentrations (1?mM) induced large, non-inactivating currents through nvTRPM2. In lower concentrations (?0.5?mM), it prevented the fast current inactivation typical for nvTRPM2 stimulated with ADPR. Both these effects were rapidly reversed after wash-out of 2-APB, in contrast to a considerable lag time of their onset. A detailed analysis of nvTRPM2 mutants with modified selectivity filter or reduced ADP-ribose sensitivity revealed that the actions of 2-APB depend on its access to the pore which is enhanced by channel opening. Moreover, access of Ca2+ to the pore is decisive which again depends on the open state of the channel. We conclude that separate regulatory processes by Ca2+ on the pore can be discriminated with the aid of 2-APB.
Project description:Transient receptor potential melastatin 2 (TRPM2) is a homotetrameric Ca2+-permeable cation channel important for the immune response, body temperature regulation, and insulin secretion, and is activated by cytosolic Ca2+ and ADP ribose (ADPR). ADPR binds to two distinct locations, formed by large N- and C-terminal cytosolic domains, respectively, of the channel protein. In invertebrate TRPM2 channels, the C-terminal site is not required for channel activity but acts as an active ADPR phosphohydrolase that cleaves the activating ligand. In vertebrate TRPM2 channels, the C-terminal site is catalytically inactive but cooperates with the N-terminal site in channel activation. The precise functional contributions to channel gating and the nucleotide selectivities of the two sites in various species have not yet been deciphered. For TRPM2 of the sea anemone Nematostella vectensis (nvTRPM2), catalytic activity is solely attributable to the C-terminal site. Here, we show that nvTRPM2 channel gating properties remain unaltered upon deletion of the C-terminal domain, indicating that the N-terminal site is single-handedly responsible for channel gating. Exploiting such functional independence of the N- and C-terminal sites, we selectively measure their affinity profiles for a series of ADPR analogues, as reflected by apparent affinities for channel activation and catalysis, respectively. Using site-directed mutagenesis, we confirm that the same N-terminal site observed in vertebrate TRPM2 channels was already present in ancient cnidarians. Finally, by characterizing the functional effects of six amino acid side chain truncations in the N-terminal site, we provide first insights into the mechanistic contributions of those side chains to TRPM2 channel gating.
Project description:The transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types. Channel activation requires binding of both ADP-ribose (ADPR) and Ca2+. The recently published TRPM2 structures from Danio rerio in the ligand-free and the ADPR/Ca2+-bound conditions represent the channel in closed and open states, which uncovered substantial tertiary and quaternary conformational rearrangements. However, it is unclear how these rearrangements are achieved within the tetrameric channel during channel gating. Here we report the cryo-electron microscopy structures of Danio rerio TRPM2 in the absence of ligands, in complex with Ca2+ alone, and with both ADPR and Ca2+, resolved to ~4.3?Å, ~3.8?Å, and ~4.2?Å, respectively. In contrast to the published results, our studies capture ligand-bound TRPM2 structures in two-fold symmetric intermediate states, offering a glimpse of the structural transitions that bridge the closed and open conformations.
Project description:The Ca(2+)-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 Ca(2+) 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 (PIP(2)) in the inner membrane leaflet. Most TRPM family channels inactivate upon prolonged stimulation in inside-out patches; this "rundown" is due to PIP(2) depletion. TRPM2 currents also run down within minutes, but the molecular mechanism of this process is unknown. Here we report that high-affinity PIP(2) binding regulates Ca(2+) sensitivity of TRPM2 activation. Nevertheless, TRPM2 inactivation is not due to PIP(2) 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/Ca(2+)-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.
Project description:Oxidants generated by activated endothelial cells are known to induce apoptosis, a pathogenic feature of vascular injury and inflammation from multiple pathogeneses. The melastatin-family transient receptor potential 2 (TRPM2) channel is an oxidant-sensitive Ca2+ permeable channel implicated in mediating apoptosis; however, the mechanisms of gating of the supranormal Ca2+ influx required for initiating of apoptosis are not understood.Here, we addressed the role of TRPM2 and its interaction with the short splice variant TRPM2 short variant (TRPM2-S) in mediating the Ca2+ entry burst required for induction of endothelial cell apoptosis.We observed that TRPM2-S was basally associated with TRPM2 in the endothelial plasmalemma, and this interaction functioned to suppress TRPM2-dependent Ca2+ gating constitutively. Reactive oxygen species production in endothelial cells or directly applying reactive oxygen species induced protein kinase C-? activation and phosphorylation of TRPM2 at Ser 39. This in turn stimulated a large entry of Ca2+ and activated the apoptosis pathway. A similar TRPM2-dependent endothelial apoptosis mechanism was seen in intact vessels. The protein kinase C-?-activated phosphoswitch opened the TRPM2 channel to allow large Ca2+ influx by releasing TRPM2-S inhibition of TRPM2, which in turn activated caspase-3 and cleaved the caspase substrate poly(ADP-ribose) polymerase.Here, we describe a fundamental mechanism by which activation of the trp superfamily TRPM2 channel induces apoptosis of endothelial cells. The signaling mechanism involves reactive oxygen species-induced protein kinase C-? activation resulting in phosphorylation of TRPM2-S that allows enhanced TRPM2-mediated gating of Ca2+ and activation of the apoptosis program. Strategies aimed at preventing the uncoupling of TRPM2-S from TRPM2 and subsequent Ca2+ gating during oxidative stress may mitigate endothelial apoptosis and its consequences in mediating vascular injury and inflammation.
Project description:Rise in plasma free fatty acids (FFAs) represents a major risk factor for obesity-induced type 2 diabetes. Saturated FFAs cause a progressive decline in insulin secretion by promoting pancreatic ?-cell death through increased production of reactive oxygen species (ROS). Recent studies have demonstrated that palmitate (a C16-FFA)-induced rise in ROS causes ?-cell death by triggering mitochondrial fragmentation, but the underlying mechanisms are unclear. Using the INS1-832/13 ?-cell line, here we demonstrate that palmitate generates the ROS required for mitochondrial fission by activating NOX (NADPH oxidase)-2. More importantly, we show that chemical inhibition, RNAi-mediated silencing and knockout of ROS-sensitive TRPM (transient receptor potential melastatin)-2 channels prevent palmitate-induced mitochondrial fission. Although TRPM2 activation affects the intracellular dynamics of Ca2+ and Zn2+, chelation of Zn2+ alone was sufficient to prevent mitochondrial fission. Consistent with the role of Zn2+, palmitate caused a rise in mitochondrial Zn2+, leading to Zn2+-dependent mitochondrial recruitment of Drp-1 (a protein that catalyses mitochondrial fission) and loss of mitochondrial membrane potential. In agreement with the previous reports, Ca2+ caused Drp-1 recruitment, but it failed to induce mitochondrial fission in the absence of Zn2+. These results indicate a novel role for Zn2+ in mitochondrial dynamics. Inhibition or knockout of TRPM2 channels in mouse islets and RNAi-mediated silencing of TRPM2 expression in human islets prevented FFA/cytokine-induced ?-cell death, findings that are consistent with the role of abnormal mitochondrial fission in cell death. To conclude, our results reveal a novel, potentially druggable signalling pathway for FFA-induced ?-cell death. The cascade involves NOX-2-dependent production of ROS, activation of TRPM2 channels, rise in mitochondrial Zn2+, Drp-1 recruitment and abnormal mitochondrial fission.
Project description:Transient potential receptor melastatin-2 (TRPM2) is a non-selective Ca(2+)-permeable cation channel of the TRPM channel subfamily and is mainly activated by intracellular adenosine diphosphate ribose (ADPR). Here we synthesized a 1-(2-nitrophenyl)ethyl caged ADPR (NPE-ADPR) and found that uncaging of NPE-ADPR efficiently stimulated Ca(2+), Mg(2+), and Zn(2+) influx in a concentration-dependent manner in intact human Jurkat T-lymphocytes. The cation influx was inhibited by inhibitors or knockdown of TRPM2. Likewise, uncaging of NPE-ADPR markedly induced cation entry in HEK 293 cells that overexpress TRPM2. As expected, high temperature increased the ability of the photolyzed NPE-ADPR to induce cation entry, whereas acidic pH inhibited. Moreover, the absence of extracellular Ca(2+) significantly inhibited Mg(2+) and Zn(2+) influx after uncaging NPE-ADPR. On the other hand, the absence of extracellular Na(+) or Mg(2+) had no effect on photolyzed NPE-ADPR induced Ca(2+) entry. Taken together, our results indicated that NPE-ADPR is a cell permeable ADPR analogue that is useful for studying TRPM2-mediated cation entry in intact cells.
Project description:Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca2+-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca2+ signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca2+ entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2'-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2'-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2'-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2'-deoxy-ATP in vitro. Thus, 2'-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.