LTRPC2 is a cation channel recently reported to be activated by adenosine diphosphate-ribose (ADP-ribose) and NAD. Since ADP-ribose can be formed from NAD and NAD is elevated during oxidative stress, we studied whole cell currents and increases in the intercellular free calcium concentration ( The long transient receptor potential channel 2 (LTRPC2) 1 is a member of the transient receptor potential (TRP) family of cation channels (1). Its function may not be confined to that of a Ca 2ϩ -permeable ion channel widely expressed in several cell types but may extend to the role of an enzyme, as has been shown for its relative TRP-phospholipase C interacting kinase (2). LTRPC2 contains a Nudix box in its C terminus (3) which is a common motif of enzymes degrading mostly nucleoside diphosphates (4). The protein NUDT9 that is homologous to the C terminus of LTRPC2 is a specific ADP-ribose pyrophosphatase degrading ADP-ribose (3). A similar function may be attributed to LTRPC2. Alternatively, the Nudix box may serve as a regulatory ADP-ribose-binding site because ADP-ribose has been shown to stimulate the channel activity of LTRPC2 (3). Therefore, ADP-ribose can be thought of as a novel second messenger regulating Ca 2ϩ influx. However, the stimuli and signaling pathways leading to elevated levels of ADP-ribose have not been elucidated in detail. ADP-ribose can be generated from cyclic ADP-ribose (5-7), an established messenger mobilizing Ca 2ϩ from ryanodine-sensitive calcium stores (8 -12). Moreover, ADP-ribose can be produced from NAD (13,14). This links ADP-ribose to the redox state of the cell and may lead to the assumption that ADP-ribose and ADP-ribose-induced Ca 2ϩ influx play a role during oxidative stress because a characteristic feature of oxidative stress is an increased ratio of NAD to NADH (15). In this context, it is of interest that NAD has been reported to be a further stimulus of LTRPC2 channels (16,17).To study the role of LTRPC2 in oxidative stress, we used an experimental model in which a strong oxidant, H 2 O 2 , was applied to LTRPC2-transfected cells. Indeed, H 2 O 2 evoked cation influx and increased [Ca 2ϩ ] i . Furthermore, we studied the effects of H 2 O 2 on splice variants of LTRPC2 identified in HL-60 cells and neutrophil granulocytes. One splice variant was activated by H 2 O 2 as the wild type but did not respond to ADPribose, in contrast to the wild type. Thus, oxidative stress leads to the activation of LTRPC2. Channel activation, however, does not need to be directly mediated by ADP-ribose. EXPERIMENTAL PROCEDURESMolecular Cloning-For cloning of LTRPC2 (formerly named TRPC7 (18)) with reverse transcriptase-polymerase chain reaction, total RNA was isolated from 1 to 2 ϫ 10 7 undifferentiated HL-60 cells using TRIzol (Invitrogen, Groningen, the Netherlands). mRNA was extracted with 15 l of Oligotex (Qiagen, Hilden, Germany). First strand cDNA synthesis was performed with 500 ng of HL-60 mRNA with Moloney murine leukemia virus reverse transcriptase (Superscript II, Invitrogen) using 500...

An early key event in the activation of neutrophil granulocytes is Ca(2+) influx. Members of the transient receptor potential (TRP) channel family may be held responsible for this. The aim of the present study is to analyse the expression pattern of TRP mRNA and identify characteristic currents unambiguously attributable to particular TRP channels. mRNA was extracted from human neutrophils, isolated by gradient centrifugation and also by magnetically labelled CD15 antibodies. The presence of mRNA was demonstrated using reverse transcriptase-PCR in neutrophils (controlled to be CD5-negative) as well as in human leukaemic cell line 60 (HL-60) cells, for the following TRP species: the long TRPC2 (LTRPC2), the vanilloid receptor 1, the vanilloid receptor-like protein 1 and epithelial Ca(2+) channels 1 and 2. TRPC6 was specific for neutrophils, whereas only in HL-60 cells were TRPC1, TRPC2, TRPC3, melastatin 1 and melastatin-related 1 found. Patch-clamp measurements in neutrophils revealed non-selective cation currents evoked by intracellular ADP-ribose and by NAD(+). Both these modes of activation have been found to be characteristic of LTRPC2. Furthermore, single-channel activity was resolved in neutrophils and it was indistinguishable from that in LTRPC2-transfected HEK-293 cells. The results provide evidence that LTRPC2 in neutrophil granulocytes forms an entry pathway for Na(+) and Ca(2+), which is regulated by ADP-ribose and the redox state.

International audienceTRPM2 is a Ca 2+}-permeable cation channel gated by ADP-ribose (ADPR) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC protocol to determine ADPR contents in perchloric acid cell extracts. The reversed phase ion-pair HPLC protocol with a Mg 2+} containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were about 5 {mu}M in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP. We furthermore analyzed intracellular concentrations of cyclic ADPR (cADPR) with a cyclase assay involving enzymatic conversion of cADPR to NAD and fluorometric determination of NAD. Intracellular cADPR concentrations were about 0.2 {mu}M and not altered by fMLP. In patch-clamp experiments, ADPR (0.1 to 100 {mu}M) was dialyzed into granulocytes to analyze its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (< 10 nM) or a high (1 {mu}M) intracellular Ca 2+} concentration. TRPM2 currents were significantly larger in high than in low Ca 2+} (e.g. -225 ± 27.1 vs. -7 ± 2.0 pA/pF at 5 {mu}M ADPR) but no currents at all were observed in the absence of ADPR (ADPR concentration <= 0.3 {mu}M). cADPR (0.1, 0.3 and 10 {mu}M) was without effect even in the presence of subthreshold ADPR (0.1 {mu}M). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca 2+}. Thus, ADPR and Ca 2+} in concert behave as a messenger system for agonist-induced influx of Ca 2+} through TRPM2 in granulocytes

Roll out the barrel: The conformation of the N‐terminal domain of a functional human voltage‐dependent anion channel (hVDAC1) in lipid bilayers has been determined (see picture; overlay of NMR model (blue) and X‐ray structure (red/gray)). Solid‐state NMR spectroscopy reveals that the N terminus assumes a well‐defined, rigid structure and that its removal induces a conformational change in the hVDAC1 β‐barrel.