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...

Depletion of intracellular calcium stores generates a signal that activates Ca2+-permeable channels in the plasma membrane. We have identified a human cDNA, TRPC1A, from a human fetal brain cDNA library. TRPC1A is homologous to the cation channels trp and trpl in Drosophila and is a splice variant of the recently identified cDNA Htrp-1. Expression of TRPC1A in CHO cells induced nonselective cation currents with similar permeabilities for Na+, Ca2+, and Cs+. The currents were activated by intracellular infusion of myo inositol 1,4,5-trisphosphate or thapsigargin. Expression of TRPC1A significantly enhanced increases in the intracellular free calcium concentration induced by Ca2+ restitution after prolonged depletion. Similar results were obtained in Sf9 cells. We conclude that TRPC1A encodes a Ca2+-permeable cation channel activated by depletion of intracellular calcium stores.

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.

We studied the role of the membrane potential in the control of the intracellular free calcium concentration ([Ca2+]i) and release of the two autacoids endothelium-derived relaxing factor (EDRF = nitric oxide) and prostaglandin I2 in endothelial cells. ATP (3 mumol/l) and bradykinin (1 nmol/l) evoked rapid increases (sixfold) in [Ca2+]i in cultured endothelial cells. [Ca2+]i remained elevated over several minutes. When the cells were depolarized, either by K+ (70-90 mmol/l) or by preincubation with the blocker of K+ channels tetraethylammonium (3 mmol/l), the initial peak of [Ca2+]i remained unaffected but [Ca2+]i returned significantly faster to resting levels, indicating a reduction in Ca2+ influx. In native, freshly isolated endothelial cells, K+ abolished increases in [Ca2+]i induced by acetylcholine (3 mumol/l). Release of EDRF in response to bradykinin (cultured cells) and acetylcholine (native cells) was inhibited by K+ (by 70%), whereas release of prostaglandin I2 was not significantly reduced. Preincubation of cultured endothelial cells with the receptor-independent stimulus thimerosal (5 mumol/l, 40 min) evoked a long-lasting release of EDRF and small elevations of [Ca2+]i (twofold) after washout of the drug. Depolarization with K+ decreased thimerosal-induced EDRF release and [Ca2+]i in a reversible manner. In patch-clamped endothelial cells, bradykinin (1 nmol/l) induced transient hyperpolarizations that were significantly prolonged by BRL 34915 (1 mumol/l), an activator of K+ channels. BRL 34915 also elicited increases in [Ca2+]i, particularly in thimerosal-stimulated endothelial cells. These effects were abolished by K+. We conclude that the initial rise in [Ca2+]i in response to receptor-binding agonists, caused by mobilization of Ca2+ from intracellular stores, activates K+ channels, thereby inducing hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)