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The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.

ABSTRACT: Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s(-1)), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 ?M), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies.

SUBMITTER: Iordanov I

PROVIDER: S-EPMC4974056 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.

Iordanov Iordan I Mihályi Csaba C Tóth Balázs B Csanády László L

eLife 20160706

Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we de ...[more]

PMID: 27383051

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Project description:Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7?kinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7?kinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7?kinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7?kinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7?kinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7?kinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7?kinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension and associated vascular and target organ damage.

Project description:RATIONALE:Obesity leads to resistant hypertension and mechanisms are poorly understood, but high plasma levels of leptin have been implicated. Leptin increases blood pressure acting both centrally in the dorsomedial hypothalamus and peripherally. Sites of the peripheral hypertensive effect of leptin have not been identified. We previously reported that leptin enhanced activity of the carotid sinus nerve, which transmits chemosensory input from the carotid bodies (CBs) to the medullary centers, and this effect was abolished by nonselective blockers of Trp (transient receptor potential) channels. We searched our mouse CB transcriptome database and found that the Trpm7 (transient receptor potential melastatin 7) channel was the most abundant Trp channel. OBJECTIVE:To examine if leptin induces hypertension acting on the CB Trpm7. METHODS AND RESULTS:C57BL/6J (n=79), leptin receptor (LepRb) deficient db/db mice (n=22), and LepRb-EGFP (n=4) mice were used. CB Trpm7 and LepRb gene expression was determined and immunohistochemistry was performed; CB glomus cells were isolated and Trpm7-like current was recorded. Blood pressure was recorded continuously in (1) leptin-treated C57BL/6J mice with intact and denervated CB; (2) leptin-treated C57BL/6J mice, which also received a nonselective Trpm7 blocker FTY720 administered systemically or topically to the CB area; (3) leptin-treated C57BL/6J mice transfected with Trpm7 small hairpin RNA to the CB, and (4) Leprb deficient obese db/db mice before and after Leprb expression in CB. Leptin receptor and Trpm7 colocalized in the CB glomus cells. Leptin induced a nonselective cation current in these cells, which was inhibited by Trpm7 blockers. Leptin induced hypertension in C57BL/6J mice, which was abolished by CB denervation, Trpm 7 blockers, and Trpm7 small hairpin RNA applied to CBs. Leprb overexpression in CB of Leprb-deficient db/db mice demethylated the Trpm7 promoter, increased Trpm7 gene expression, and induced hypertension. CONCLUSIONS:We conclude that leptin induces hypertension acting on Trmp7 in CB, which opens horizons for new therapy.

Dataset Information

The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.

Publications

The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.

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