Anne-Laure Perraud

ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology

Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

Regulation of Vertebrate Cellular Mg2+ Homeostasis by TRPM7

TRPM7 is a polypeptide with intrinsic ion channel and protein kinase domains whose targeted deletion causes cells to experience growth arrest within 24 hr and eventually die. Here, we show that while TRPM7s kinase domain is not essential for activation of its channel, a functional coupling exists such that structural alterations of the kinase domain alter the sensitivity of channel activation to Mg(2+). Investigation of the relationship between Mg(2+) and the cell biological role of TRPM7 revealed that TRPM7-deficient cells become Mg(2+) deficient, that both the viability and proliferation of TRPM7-deficient cells are rescued by supplementation of extracellular Mg(2+), and that the capacity of heterologously expressed TRPM7 mutants to complement TRPM7 deficiency correlates with their sensitivity to Mg(2+). Overall, our results indicate that TRPM7 has a central role in Mg(2+) homeostasis as a Mg(2+) uptake pathway regulated through a functional coupling between its channel and kinase domains.

TRPM4 Is a Ca2+-Activated Nonselective Cation Channel Mediating Cell Membrane Depolarization

Calcium-activated nonselective (CAN) cation channels are expressed in various excitable and nonexcitable cells supporting important cellular responses such as neuronal bursting activity, fluid secretion, and cardiac rhythmicity. We have cloned and characterized a second form of TRPM4, TRPM4b, a member of the TRP channel family, as a molecular candidate of a CAN channel. TRPM4b encodes a cation channel of 25 pS unitary conductance that is directly activated by [Ca2+]i with an apparent K(D) of approximately 400 nM. It conducts monovalent cations such as Na+ and K+ without significant permeation of Ca2+. TRPM4b is activated following receptor-mediated Ca2+ mobilization, representing a regulatory mechanism that controls the magnitude of Ca2+ influx by modulating the membrane potential and, with it, the driving force for Ca2+ entry through other Ca2+-permeable pathways.

The Channel Kinases TRPM6 and TRPM7 Are Functionally Nonredundant

TRPM7 and its closest homologue, TRPM6, are the only known fusions of an ion channel pore with a kinase domain. Deletion of TRPM7 in DT40 B-lymphocytes causes growth arrest, Mg2+ deficiency, and cell death within 24–48 h. Amazingly, in analogy to TRPM6-deficient patients who can live a normal life if provided with a Mg2+-rich diet, TRPM7-deficient DT40 B-lymphocytes show wild type cell growth if supplied with 5–10 mm Mg2+ concentrations in their extracellular medium. Here we have investigated the functional relationship between TRPM6 and TRPM7. We show that TRPM7 deficiency in DT40 cells cannot be complemented by heterologously expressed TRPM6. Nevertheless, both channels can influence each others biological activity. Our data demonstrate that TRPM6 requires TRPM7 for surface expression in HEK-293 cells and also that TRPM6 is capable of cross-phosphorylating TRPM7 as assessed using a phosphothreonine-specific antibody but not vice versa. TRPM6 and TRPM7 coexpression studies in DT40 B-cells indicate that TRPM6 can modulate TRPM7 function. In conclusion, although TRPM6 and TRPM7 are closely related and deficiency in either one of these molecules severely affects Mg2+ homeostasis regulation, TRPM6 and TRPM7 do not appear to be functionally redundant but rather two unique and essential components of vertebrate ion homeostasis regulation.

MPYS Is Required for IFN Response Factor 3 Activation and Type I IFN Production in the Response of Cultured Phagocytes to Bacterial Second Messengers Cyclic-di-AMP and Cyclic-di-GMP

Cyclic-di-GMP and cyclic-di-AMP are second messengers produced by bacteria and influence bacterial cell survival, differentiation, colonization, biofilm formation, virulence, and bacteria–host interactions. In this study, we show that in both RAW264.7 macrophage cells and primary bone marrow-derived macrophages, the production of IFN-β and IL-6, but not TNF, in response to cyclic-di-AMP and cyclic-di-GMP requires MPYS (also known as STING, MITA, and TMEM173). Furthermore, expression of MPYS was required for IFN response factor 3 but not NF-κB activation in response to these bacterial metabolites. We also confirm that MPYS is required for type I IFN production by cultured macrophages infected with the intracellular pathogens Listeria monocytogenes and Francisella tularensis. However, during systemic infection with either pathogen, MPYS deficiency did not impact bacterial burdens in infected spleens. Serum IFN-β and IL-6 concentrations in the infected control and MPYS−/− mice were also similar at 24 h postinfection, suggesting that these pathogens stimulate MPYS-independent cytokine production during in vivo infection. Our findings indicate that bifurcating MPYS-dependent and -independent pathways mediate sensing of cytosolic bacterial infections.

TRPM2 Ca2+ permeable cation channels: from gene to biological function.

TRPM2 is a recently identified TRPM family cation channel which is unique among known ion channels in that it contains a C-terminal domain which is homologous to the NUDT9 ADP-ribose hydrolase and possesses intrinsic ADP-ribose hydrolase activity. Here, available information on the TRPM2 gene, transcripts, predicted protein products, and assembled multimeric channels is comprehensively reviewed and synthesized to highlight important areas for future work and provide insight into potential biological function(s) of TRPM2 channels.

Metabolite of SIR2 reaction modulates TRPM2 ion channel

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins are protein deacetylases that regulate gene expression, apoptosis, metabolism, and aging. These NAD+-dependent enzymes catalyze a reaction in which the acetyl group from substrate is transferred to the ADP-ribose portion of NAD+ to form deacetylated product, nicotinamide, and the metabolite OAADPr, whose functions remain elusive. Using cell-based assays and RNA interference, we show that puromycin-induced cell death is greatly diminished by nicotinamide (a potent sirtuin inhibitor), and by decreased expression of sirtuins SIRT2 and SIRT3. Furthermore, we demonstrate using channel current recordings and binding assays that OAADPr directly binds to the cytoplasmic domain of TRPM2 and activates the TRPM2 channel. ADP-ribose binds TRPM2 with similarly affinity, whereas NAD+ displays almost negligible binding. These studies provide the first evidence for the potential role of sirtuin-generated OAADPr in TRPM2 channel gating.

Transient Receptor Potential Melastatin 2 (TRPM2) ion channel is required for innate immunity against Listeria monocytogenes

The generation of reactive oxygen species (ROS) is inherent to immune responses. ROS are crucially involved in host defense against pathogens by promoting bacterial killing, but also as signaling agents coordinating the production of cytokines. Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca2+-permeable channel gated via binding of ADP-ribose, a metabolite formed under conditions of cellular exposure to ROS. Here, we show that TRPM2-deficient mice are extremely susceptible to infection with Listeria monocytogenes (Lm), exhibiting an inefficient innate immune response. In a comparison with IFNγR-deficient mice, TRPM2−/− mice shared similar features of uncontrolled bacterial replication and reduced levels of inducible (i)NOS-expressing monocytes, but had intact IFNγ responsiveness. In contrast, we found that levels of cytokines IL-12 and IFNγ were diminished in TRPM2−/− mice following Lm infection, which correlated with their reduced innate activation. Moreover, TRPM2−/− mice displayed a higher degree of susceptibility than IL-12–unresponsive mice, and supplementation with recombinant IFNγ was sufficient to reverse the unrestrained bacterial growth and ultimately the lethal phenotype of Lm-infected TRPM2−/− mice. The severity of listeriosis we observed in TRPM2−/− mice has not been reported for any other ion channel. These findings establish an unsuspected role for ADP-ribose and ROS-mediated cation flux for innate immunity, opening up unique possibilities for immunomodulatory intervention through TRPM2.

Altered functional properties of a TRPM2 variant in Guamanian ALS and PD

Two related neurodegenerative disorders, Western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism–dementia (PD), originally occurred at a high incidence on Guam, in the Kii peninsula of Japan, and in southern West New Guinea more than 50 years ago. These three foci shared a unique mineral environment characterized by the presence of severely low levels of Ca2+ and Mg2+, coupled with high levels of bioavailable transition metals in the soil and drinking water. Epidemiological studies suggest that genetic factors also contribute to the etiology of these disorders. Here, we report that a variant of the transient receptor potential melastatin 2 (TRPM2) gene may confer susceptibility to these diseases. TRPM2 encodes a calcium-permeable cation channel highly expressed in the brain that has been implicated in mediating cell death induced by oxidants. We found a heterozygous variant of TRPM2 in a subset of Guamanian ALS (ALS-G) and PD (PD-G) cases. This variant, TRPM2P1018L, produces a missense change in the channel protein whereby proline 1018 (Pro1018) is replaced by leucine (Leu1018). Functional studies revealed that, unlike WT TRPM2, P1018L channels inactivate. Our results suggest that the ability of TRPM2 to maintain sustained ion influx is a physiologically important function and that its disruption may, under certain conditions, contribute to disease states.

The TRPM2 ion channel, an oxidative stress and metabolic sensor regulating innate immunity and inflammation

TRPM2 (transient receptor potential melastatin 2) is the unique fusion of a Ca2+-permeable pore with an enzymatic domain that binds the NAD+-metabolite ADP-ribose (ADPR), resulting in channel opening. ADPR formation is a metabolic corollary of cellular stress, but can also be elicited enzymatically through NAD glycohydrolases like CD38. TRPM2 thus functions as a metabolic and oxidative stress sensor and translates this information into ion fluxes that can affect Ca2+ signaling and the membrane potential. TRPM2 is strongly represented in immune cells of the phagocytic lineage, themselves professional generators of oxidants. The recent characterization of TRPM2-deficient mouse models has revealed the involvement of this channel in various aspects of immunity. Monocytes lacking TRPM2 show reduced production of the CXCL2 chemokine, resulting in diminished neutrophilic influx to the colon in chemically induced colitis, and thus protection against tissue ulceration in TRPM2−/− mice. However, the insufficient production of proinflammatory cytokines leads to high morbidity and lethality of the TRPM2−/− mice following infection with the bacterial pathogen Listeria monocytogenes. In the context of endotoxin-induced pulmonary inflammation, TRPM2’s absence was found to promote inflammation and ROS production. TRPM2 acts thereby as a negative feedback loop by interfering through membrane depolarization with ROS generation by NADPH oxidases. In dendritic cells, TRPM2 is a lysosomal Ca2+-release channel that promotes chemokine responsiveness and cell migration, which is reminiscent of CD38-mediated functions. The discovery of TRPM2 has unveiled an unsuspected signaling pathway and established ADPR as a novel second messenger. Understanding TRPM2’s complex involvement in inflammation is crucial to evaluating the potential of manipulating TRPM2 activity and ADPR metabolism for therapeutic intervention.