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Scientific reports 9(1), 19224 (2019). doi:10.1038/s41598-019-55232-5, Published by Macmillan Publishers Limited, part of Springer Nature, [London]

PLoS one 11(6), e0158060 (2016). doi:10.1371/journal.pone.0158060, Published by PLoS, Lawrence, Kan.

The closely related cation channels TRPM2 and TRPM8 show completely different requirements for stimulation and are regulated by Ca(2+) in an opposite manner. TRPM8 is basically gated in a voltage-dependent process enhanced by cold temperatures and cooling compounds such as menthol and icilin. The putative S4 voltage sensor of TRPM8 is closely similar to that of TRPM2, which, however, is mostly devoid of voltage sensitivity. To gain insight into principal interactions of critical channel domains during the gating process, we created chimeras in which the entire S5-pore-S6 domains were reciprocally exchanged. The chimera M2-M8P (i.e. TRPM2 with the pore of TRPM8) responded to ADP-ribose and hydrogen peroxide and was regulated by extracellular and intracellular Ca(2+) as was wild-type TRPM2. Single-channel recordings revealed the characteristic pattern of TRPM2 with extremely long open times. Only at far-negative membrane potentials (-120 to -140 mV) did differences become apparent because currents were reduced by hyperpolarization in M2-M8P but not in TRPM2. The reciprocal chimera, M8-M2P, showed currents after stimulation with high concentrations of menthol and icilin, but these currents were only slightly larger than in controls. The transfer of the NUDT9 domain to the C terminus of TRPM8 produced a channel sensitive to cold, menthol, or icilin but insensitive to ADP-ribose or hydrogen peroxide. We conclude that the gating processes in TRPM2 and TRPM8 differ in their requirements for specific structures within the pore. Moreover, the regulation by extracellular and intracellular Ca(2+) and the single-channel properties in TRPM2 are not determined by the S5-pore-S6 region.

A pharmacological characterization of Ca(2+) influx pathways in neutrophil granulocytes is problematic because of the lack of specific inhibitors. The activation of transient receptor potential cation channel, subfamily M, member 2 (TRPM2) channels by intracellular adenosine diphosphate ribose (ADPR), well characterized in neutrophils, is reportedly inhibited by 8-bromo-ADPR (8Br-ADPR). TRPM2 is blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA) interfering with the pore, but ACA is as well effective on other transient receptor potential channels, especially transient receptor potential canonical (TRPC) channels. We wished to analyze whether ACA and 8Br-ADPR were suitable probes to demonstrate that different Ca(2+) entry pathways are activated in human neutrophil granulocytes by the receptor-dependent stimuli N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and platelet-activating factor (PAF) and the receptor-independent thapsigargin. Ca(2+)-influx-related increases in [Ca(2+)](i) were calculated by comparing aliquots of fluo-3-loaded neutrophils in the presence and absence of extracellular Ca(2+). Moreover, Mn(2+) quenching was used in fura-2-loaded cells. We compared 8Br-ADPR with ACA. 8Br-ADPR was exclusively effective when Ca(2+) influx (or Mn(2+) quenching) was induced by fMLP; it did not affect influx when PAF or thapsigargin was the stimulus. ACA inhibited Ca(2+) influx significantly more strongly when this was induced by PAF than by fMLP. Moreover, it reduced thapsigargin-induced Ca(2+) influx. The contribution of TRPM2 to Ca(2+) influx in neutrophils strongly depends on the stimulus; it is sizeable in the case of fMLP and minimal in the case of PAF. PAF induces Ca(2+) entry pathways different from TRPM2; the inhibition by ACA suggests the contribution of channels of the TRPC family.

Type 2 melastatin-related transient receptor potential channel (TRPM2), a member of the melastatin-related TRP (transient receptor potential) subfamily is a Ca(2+)-permeable channel activated by hydrogen peroxide (H(2)O(2)). We have investigated the role of TRPM2 channels in mediating the H(2)O(2)-induced increase in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in insulin-secreting cells. In fura-2 loaded INS-1E cells, a widely used model of beta-cells, and in human beta-cells, H(2)O(2) increased [Ca(2+)](i), in the presence of 3 mM glucose, by inducing Ca(2+) influx across the plasma membrane. H(2)O(2)-induced Ca(2+) influx was not blocked by nimodipine, a blocker of the L-type voltage-gated Ca(2+) channels nor by 2-aminoethoxydiphenyl borate, a blocker of several TRP channels and store-operated channels, but it was completely blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA), a potent inhibitor of TRPM2. Adenosine diphosphate phosphate ribose, a specific activator of TRPM2 channel and H(2)O(2), induced inward cation currents that were blocked by ACA. Western blot using antibodies directed to the epitopes on the N-terminal and on the C-terminal parts of TRPM2 identified the full length TRPM2 (TRPM2-L), and the C-terminally truncated TRPM2 (TRPM2-S) in human islets. We conclude that functional TRPM2 channels mediate H(2)O(2)-induced Ca(2+) entry in beta-cells, a process potently inhibited by ACA.

In the dysfunctional splice variant TRPM2-DeltaN, a stretch of 20 amino acids (aa 537-556) is missing within the N-terminal cytosolic tail of the cation channel TRPM2. The DeltaN-stretch overlaps with two IQ-like calmodulin-binding domains. Moreover, it contains two PxxP motifs implicated in protein-protein interactions. Here, we constructed variants to test whether any of these motifs may explain why TRPM2-DeltaN does not respond to stimulation with either ADP ribose or hydrogen peroxide. Each of the two IQ-motifs could be removed without loss of channel function. Similarly, deletion of either one or both PxxP motifs had no effect. Moreover, the single point mutation D543E associated with bipolar disorder does not change the activation of TRPM2. We conclude that no functional role can be attributed to any of the structural motifs within the DeltaN-stretch that may be a spacer segment for other functional sites in the N terminus.

TRPM2 and TRPM8, closely related members of the transient receptor potential (TRP) family, are cation channels activated by quite different mechanisms. Their transmembrane segments S5 and S6 are highly conserved. To identify common structures in S5 and S6 that govern interaction with the pore, we created a chimera in which the S5-pore-S6 region of TRPM8 was inserted into TRPM2, along with a lysine at each transition site. Currents through this chimera were induced by ADP-ribose (ADPR) in cooperation with Ca(2+). In contrast to wild-type TRPM2 channels, currents through the chimera were carried by Cl(-), as demonstrated in ion substitution experiments using the cation N-methyl-D-glucamine (NMDG) and the anion glutamate. Extracellular NMDG had no effects. The substitution of either intracellular or extracellular Cl(-) with glutamate shifted the reversal potential, decreased the current amplitude and induced a voltage-dependent block relieved by depolarization. The lysine in S6 was responsible for the anion selectivity; insertion of a lysine into corresponding sites within S6 of either TRPM2 or TRPM8 created anion channels that were activated by ADPR (TRPM2 I1045K) or by cold temperatures (TRPM8 V976K). The positive charge of the lysine was decisive for the glutamate block because the mutant TRPM2 I1045H displayed cation currents that were blocked at acidic but not alkaline intracellular pH values. We conclude that the distal part of S6 is crucial for the discrimination of charge. Because of the high homology of S6 in the whole TRP family, this new role of S6 may apply to further TRP channels.

TRPM2 (transient receptor potential melastatin 2) is a Ca2+-permeable cation channel gated by ADPR (ADP-ribose) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC method to determine ADPR contents in HClO4 cell extracts. The reversed-phase ion-pair HPLC method with an Mg2+-containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were approx. 5 muM in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP (N-formylmethionyl-leucylphenylalanine). We furthermore determined intracellular concentrations of cADPR (cyclic ADPR) with a cyclase assay involving enzymatic conversion of cADPR into NAD+ and fluorimetric determination of NAD+. Intracellular cADPR concentrations were approx. 0.2 microM and not altered by fMLP. In patch-clamp experiments, ADPR (0.1-100 microM) was dialysed into granulocytes to analyse its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (10 nM) or a high (1 microM) intracellular Ca2+ concentration. TRPM2 currents were significantly larger at high than at low [Ca2+] (e.g. -225+/-27.1 versus -7+/-2.0 pA/pF at 5 muM ADPR), but no currents at all were observed in the absence of ADPR (ADPR concentrationor =0.3 microM). cADPR (0.1, 0.3 and 10 microM) was without effect even in the presence of subthreshold ADPR (0.1 microM). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca2+. Thus ADPR and Ca2+ in concert behave as a messenger system for agonist-induced influx of Ca2+ through TRPM2 in granulocytes.

A unique functional property within the transient receptor potential (TRP) family of cation channels is the gating of TRP (melastatin) 2 (TRPM2) channels by ADP-ribose (ADPR). ADPR binds to the intracellular C-terminal tail of TRPM2, a domain that shows homology to enzymes with pyrophosphatase activity. Cytosolic Ca(2+) enhances TRPM2 gating by ADPR; ADPR and Ca(2+) in concert may be an important messenger system mediating Ca(2+) influx. Other stimuli of TRPM2 include NAD and H(2)O(2) and cyclic ADPR, which may act synergistically with ADPR. H(2)O(2), an experimental paradigm of oxidative stress, may also induce the formation of ADPR in the nucleus or mitochondria. In this review, we summarize the gating properties of TRPM2 and the proposed pathways of channel activation in vivo. TRPM2 is likely to be a key player in several signalling pathways, mediating cell death in response to oxidative stress or in reperfusion injury. Moreover, it plays a decisive role in experimentally induced diabetes mellitus and in the activation of leukocytes.

TRPM2 channels play an important role in the activation process of neutrophil granulocytes. One mechanism of TRPM2 channel gating is the binding of intracellular ADP ribose (ADPR) to the Nudix box domain in the C-terminal tail of TRPM2. Intracellular Ca(2+), although not an activator of TRPM2 by its own, significantly enhances TRPM2 gating by ADPR. Stimulation of neutrophil granulocytes with the chemoattractant peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) induces release of Ca(2+) ions from intracellular stores which in cooperation with endogenous ADPR levels enable Ca(2+) influx through TRPM2. Stimulation of the ectoenzyme CD38, a membrane-associated glycohydrolase with ADPR as main product, and uptake of ADPR into the cell may contribute to the effects of fMLP. Inhibition of ADPR production, of uptake and of binding to TRPM2 are all potential pharmacological principles by which a modulation of neutrophil function may become possible in future.

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 ([Ca2+]i) in long transient receptor potential channel 2(LTRPC2)-transfected HEK 293 cells after stimulation with hydrogen peroxide (H2O2). Cation currents carried by monovalent cations and Ca2+ were induced by H2O2 (5 mm in the bath solution) as well as by intracellular ADP-ribose (0.3 mm in the pipette solution) but not by NAD (1 mm). H2O2-induced currents developed slowly after a characteristic delay of 3–6 min and receded after wash-out of H2O2. [Ca2+]i was rapidly increased by H2O2 inLTRPC2-transfected cells as well as in control cells; however, in LTRPC2-transfected cells, H2O2 evoked a second delayed rise in [Ca2+]i. A splice variant of LTRPC2 with a deletion in the C terminus (amino acids 1292–1325) was identified in neutrophil granulocytes. This variant was stimulated by H2O2 as the wild type. However, it did not respond to ADP-ribose. We conclude that activation of LTRPC2 by H2O2 is independent of ADP-ribose and that LTRPC2 may mediate the influx of Na+ and Ca2+during oxidative stress, such as the respiratory burst in granulocytes.

The lanthanide ions La(3+) and Gd(3+) block Ca(2+)-permeable cation channels and have been used as important tools to characterize channels of the transient receptor potential (TRP) family. However, widely different concentrations of La(3+) and Gd(3+) have reportedly been required for block of TRP3 channels in various expression systems. The present study provides a possible explanation for this discrepancy. After overexpression of TRP3 in Chinese hamster ovary cells, whole-cell currents through TRP3 were reversibly inhibited by La(3+) with an EC(50) of 4 microm. For comparison, the organic blocker SKF96365 required an EC(50) of 8 microm. Gd(3+) blocked with an EC(50) of 0.1 microm, but this block was slow in onset and was not reversible after wash-out. When the two lanthanides were added to the cytosolic side of inside-out patches, block was achieved with considerably lower concentrations (EC(50) for La(3+), 0.02 microm; EC(50) for Gd(3+), 0.02 microm). Uptake of La(3+) into the cytosol of Chinese hamster ovary cells was demonstrated with intracellular fura-2. We conclude that lanthanides block TRP3 more potently from the cytosolic than from the extracellular side of the plasma membrane and that uptake of lanthanides will largely affect the apparent EC(50) values after extracellular application.

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.

For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of voltage-dependent cation channels, among them TRPM8. Moreover, they are also conserved in the closely related TRPM2 channel, which is essentially voltage-independent. To analyze the differential roles of the motif for the voltage-dependent and voltage-independent gating, we performed reciprocal replacements of the asparagine and aspartate within the S3 motif in both channels, following the proposed idea that specific electrostatic interactions with other domains take place during gating. Wild-type and mutant channels were heterologeously expressed in HEK-293 cells and channel function was analyzed by whole-cell patch-clamp analysis as well as by Ca(2+)-imaging. Additionally, the expression of the channels in the plasma membrane was tested by Western blot analysis, in part after biotinylation. For the mutations of TRPM8, responses to menthol were only compromised if also the expression of the glycosylated channel isoform was prevented. In contrast, responses to cold were consistently and significantly attenuated but not completely abolished. For TRPM2, surface expression was not significantly affected by any of the mutations but channel function was only retained in one variant. Remarkably, this was the variant of which the corresponding mutation in TRPM8 exerted the most negative effects both on channel function and expression. Furthermore, we performed an exchange of the inner pair of residues of the N-x-x-D motif between the two channels, which proved deleterious for the functional expression of TRPM8 but ineffective on TRPM2. In conclusion, the N-x-x-D motif plays specific roles in TRPM8 and TRPM2, reflecting different requirements for voltage-dependent and voltage-independent channel gating.

TRPM8 is a cation channel activated by cold temperatures and the chemical stimuli menthol and icilin. Both compounds use different mechanisms of current activation; amino acid residues within the S2-S3 linker have been identified critical for current activation by icilin but not by menthol. Current decline in the course of menthol stimulation reflects Ca(2+)-dependent desensitization attributed to phosphatidylinositol 4,5-bisphosphate depletion. Carboxyamide derivatives chemically resembling menthol have been described as activators of TRPM8 analogous to icilin. Our aim was a detailed analysis of whether differences exist between all these substances with respect to their activation and inactivation of currents. We studied wild-type TRPM8 as well as an s3-TRPM8 mutant with mutations in the S2-S3 linker region that could not be activated by icilin. Menthol and menthol derivatives behaved indistinguishable in evoking currents through both channels in a Ca(2+)-independent manner as well as inducing Ca(2+)-dependent desensitization. Icilin, in contrast, activated currents only in wild type TRPM8 and in the presence of Ca(2+). Moreover, it completely reversed currents induced by menthol, menthol derivatives, and cold temperatures in wild type TRPM8 and s3-TRPM8; this current inhibition was independent of Ca(2+). Finally, icilin suppressed current activation by the other agonists. None of the inhibiting effects of icilin occurred in the cation channel TRPA1 that is also stimulated by both menthol and icilin. Thus, icilin specifically inhibits TRPM8 independently of its interaction site within the S2-S3 linker through a process distinct from desensitization.

The aim of the present study was to analyse whether an increase in the intracellular free Ca2+ concentration ([Ca2+]i) plays a role as a signal mediating synthesis of nitric oxide (NO) in bone-marrow-derived macrophages, either by stimulating induction of NO synthase or by regulating the activity of the enzyme. Therefore we compared the effects of various synthetic analogues of bacterial lipopeptide and of lipopolysaccharide (LPS) on NO production (assessed as nitrite formation during an incubation for 24 h) and on [Ca2+]i [measured with the fluorescent probe indo-1 (1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2- 2-(2′-amino-5′-methylphenoxy)ethane-NNN′N′-tetra-acetic acid)]. Strongly dissociating effects were evoked on nitrite formation and on [Ca2+]i by the stimuli. LPS was preferentially effective on nitrite formation, whereas the Ca2+ ionophore ionomycin and AlF3 induced increases only in [Ca2+]i. The lipopeptides N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinylalanylglycine, N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-(R)-cysteinylseryl-lysyl-lysyl-lysine and (S)-(1,2- dicarboxyhexadecyl)ethyl-N-palmitoylcysteinylseryl-lysyl-lys yl-lysine stimulated both parameters, but the maximal effects on nitrite formation and the shape of the dose-response curves did not parallel the effects on [Ca2+]i. Reduction of extracellular Ca2+ with EGTA significantly inhibited increases in [Ca2+]i, but did not change nitrite formation. Furthermore, NO synthesis in the cytosolic fraction of stimulated macrophages was not affected by Ca2+ over the concentration range 10 nM-2 microM. We conclude that increases in [Ca2+]i are not required for NO production in bone-marrow-derived macrophages. Thus the cellular regulation of NO production strikingly differs from that in the vascular endothelium, brain and adrenal gland.

A melastatin-like transient receptor potential 2 (TRPM2) channel is activated in concert with Ca2+ by intracellular adenosine diphosphoribose (ADPR) binding to the channel's enzyme Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD+) and hydrogen peroxide (H2O2) although the mechanisms remain unknown. Hence, we tested the effects of ADPR, NAD+ and H2O2 on the activation of TRPM2 currents in transfected Chinese hamster ovary (CHO) cells. The CHO cells were transfected with cDNA coding for TRPM2. The intracellular solution used EDTA (10 mM) as a chelator for Ca2+ and heavy metal ions. Moreover, we balanced the intracellular Ca2+ concentration at 1 microM. H2O2 (10 mM) in the bath chamber was extracellularly added although ADPR (0.3 mM) and NAD+ (1 mM) in pipette solution were intracellularly added. Using these conditions, the channel currents were evoked by the three stimulators. The time course of ADPR, NAD+ and H2O2 effects was characterized by a delay of 0.6, 3.0 min and 2-5 min, respectively and a slow current induction reached a clear plateau with ADPR and NAD+ although H2O2 currents continued to gain in amplitude over at least 15 min and it did not reach a clear plateau in many experiments. Furthermore, H2O2-induced a single-channel conductance in the current study; the first time that this has been resolved in CHO. The conductance of ADPR and H2O2 was 48.80 pS and 39.14 pS, respectively and the cells seem to be separately activated by ADPR and H2O2. In conclusion, we observed further support for a calcium influx pathway regulated separately by oxidative stress and ADPR in TRPM2 channels in transfected cells. A second novel result of the present study was that the TRPM2 channels were constitutionally activated by H2O2.

The melastatin-related transient receptor potential channel TRPM2 is a plasma membrane Ca2+-permeable cation channel that is activated by hydrogen peroxide (H2O2) as a consequence of oxidative stress although the channel activation by H2O2 appears to represent a cell-specific process in cells with endogenous expression of TRPM2. Flufenamic acid (FA) is a non-steroidal anti-inflammatory compound. Whether H2O2 activates or FA inhibits TRPM2 channels in Chinese hamster ovary (CHO) cell is currently unknown. Due to lack of known antogonists of this channel, we demonstrate in CHO cells that FA inhibits TRPM2 activated by extracellular H2O2. CHO cells were transfected with cDNA coding for TRPM2. Cells were studied with the conventional whole-cell patch clamp technique. The intracellular solution used EDTA (10 mM) as chelator for Ca2+ and heavy metal ions. H2O2 (10 mM) and FA (0.1 mM) were applied extracellularly. Non-selective cation currents were consistently induced by H2O2. The time cause of H2O2 effects was characterized by a delay of 2–5 min and a slow current induction to reach a plateau. The H2O2- induced inward current was effectively inhibited by 0.1 mM FA applied extracellularly. In conclusion, we have demonstrated that FA is an effective antogonist of TRPM2 channels and H2O2activated currents in CHO cells. FA in CHO cells may be considered, at best, a starting point for the development of TRPM2 channel blockers. Copyright © 2006 John Wiley & Sons, Ltd.

TRPM2 is a cation channel unique within the transient receptor potential family because of its gating by ADP-ribose (ADPR). ADPR gating is enabled by a cytosolic C-terminal Nudix box sequence motif embedded into a region homologous to the NUDT9 ADPR pyrophosphatase. A recently discovered splice variant of TRPM2 (TRPM2-DeltaC) lacks 34 amino acid residues in the NUDT9 domain and is insensitive to ADPR. To analyze in detail which parts of the deleted sequence (DeltaC-stretch) are critical for ADPR gating, we tested mutants that lacked 19, 25, and 29 amino acid residues in the N-terminal part or had amino acid residues substituted in the remaining C-terminal part of the DeltaC-stretch. All of these mutants displayed typical ADPR-induced currents. However, the deletion or substitution of the amino acid residue Asn-1326 immediately downstream of the DeltaC-stretch abrogated ADPR gating. We furthermore analyzed the mutation I1405E/L1406F in the Nudix box of TRPM2, because a considerably decreased AD-PRase activity of the TRPM2 NUDT9-H protein in comparison to the NUDT9 pyrophosphatase has been attributed to the reverse exchange EF --> IL. The I1405E/L1406F variant of TRPM2 failed to respond to ADPR even at concentrations up to 10 mM. We concluded that the DeltaC-stretch contains no individual amino acid residues essential for ADPR gating but may act as a spacer segment stabilizing a conformation necessary for the essential residue Asn-1326 to interact with other channel regions. Enhancing the enzymatic activity of the Nudix box abolishes the ADPR gating of TRPM2, pointing to the requirement of prolonged binding rather than degradation.

An early key event in the activation of neutrophil granulocytes is Ca2+ 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 Ca2+ 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 Ca2+, which is regulated by ADP-ribose and the redox state.

Receptor-mediated increases in the cytosolic free calcium ion concentration in most mammalian cells result from mobilization of Ca2+ from intracellular stores as well as transmembrane Ca2+ influx. Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores by opening a Ca(2+)-permeable channel in the endoplasmic reticulum. But the mechanism and regulation of Ca2+ entry into nonexcitable cells has remained elusive because the entry pathway has not been defined. Here we characterize a novel inositol 1,3,4,5-tetrakisphosphate (InsP4) and Ca(2+)-sensitive Ca(2+)-permeable channel in endothelial cells. We find that InsP4, which induces Ca2+ influx into acinar cells, enhances the activity of the Ca(2+)-permeable channel when exposed to the intracellular surface of endothelial cell inside-out patches. Our results suggest a molecular mechanism which is likely to be important for receptor-mediated Ca2+ entry.

The present study was undertaken to define the route of Ca2+ used for refilling of intracellular Ca2+ stores in endothelial cells. Ca2+ stores, after emptying with bradykinin in Ca2+ free solution and termination of the stimulation with the bradykinin antagonist, Hoe 140, were allowed to refill by addition of Ca2+. Refilling was prevented by 2,5-di(tert-butyl)-1,4-benzohydroquinone (BuBHQ), an inhibitor of microsomal Ca2+ sequestration. BuBHQ induced large increases in the cytosolic Ca2+ concentration during the refilling phase. This finding is not compatible with a model proposing Ca2+ uptake into the stores directly from the extracellular space but provides evidence for uptake from the cytosolic compartment.

The Drosophila melanogaster gene product TRPL (transient receptor potential-like) is a Ca2+-permeable cation channel that contributes to the light-induced Ca2+ entry in Drosophila photoreceptors and bears homology to several recently cloned mammalian channels. Intracellular Ca2+ has been implicated to stimulate TRPL channels. This constitutes a potentially dangerous mechanism that may lead to Ca2+ overload. Therefore, we studied whether TRPL channels, like other Ca2+-permeable channels, are inhibited by intracellular Ca2+ concentrations in the micromolar range and whether this effect is mediated by calmodulin. In Sf9 cells expressing the TRPL gene along with histamine H1 receptors after infection with baculoviruses containing the corresponding complementary DNA, histamine-induced TRPL currents were inhibited by intracellular Ca2+ with an IC50 of 2.3 microM. Moreover, TRPL currents were reversibly attenuated by a preceding hyperpolarization. This attenuation reflected the action of an increased Ca2+ influx, since it was abolished in the absence of extracellular Ca2+ and enhanced by raising extracellular Ca2+ to 20 mM. Finally, the activity of TRPL channels in inside-out patches was reversibly inhibited by raising the Ca2+ concentration on the cytosolic side of the patches to 10-50 microM. Addition of calmodulin or the calmodulin inhibitor calmidazolium did not modify the inhibition of the TRPL by Ca2+. We conclude that high intracellular Ca2+ concentrations inhibit the TRPL, but no evidence was found for the requirement of calmodulin. This mechanism makes Ca2+ influx through the TRPL self-limiting. Furthermore, the TRPL may allow one to study the structural requirements for channel regulation by Ca2+.

We investigated the molecular mechanisms whereby Ca2+ enters the endothelial cytosol and regulates endothelial nitric oxide synthesis L-arginine-dependent nitric oxide synthesis by isolated endothelial cytosol as quantified by activation of a purified soluble guanylate cyclase was concentration-dependently enhanced by free Ca2+ (EC50 0.3 μM). The Ca2+-dependent activation was inhibited by the calmodulin antagonists mastoparan, melittin, and calcineurin (IC50 450, 350, and 60 nM, respectively) in a calmodulin-reversible manner. After removal of endogenous calmodulin the Ca2+-dependency of endothelial NO synthase was lost, but could be reconstituted with exogenous calmodulin. The results indicate that Ca2+-calmodulin directly activates the endothelial nitric oxide synthase, thereby transducing agonist-induced increases in intracellular free Ca2+ concentration to nitric oxide formation from L-arginine, K+-induced depolarization of the endothelial cells markedly inhibited the sustained, but not initial phase of the intracellular Ca2+ response to bradykinin, indicating that K+-induced depolarization depresses the transmembrane Ca2+ influx. On the contrary, the K+ channel activator Hoe 234 which elicits hyperpolarization of the endothelial cell membrane, augmented the sustained phase of the agonist-induced intracellular Ca2+ signal, but not the resting intracellular Ca2+ level. The effects of K+ and Hoe 234 on the agonist-induced Ca2+-response were reflected by corresponding changes in agonist-induced EDRF/NO release. From these data, we suggest that the endothelial membrane potential may play an important role for the extent of agonist-induced Ca2+ influx and, thereby, the endothelial EDRF/NO synthesis.

Ca2+ influx into stimulated endothelial cells is attenuated by depolarization. We hypothesized that Ca2+ influx is driven by the membrane potential and may be enhanced by hyperpolarizing drugs like activators of K+ channels. Therefore we studied the effects of pinacidil, cromakalim, and cicletanine on membrane currents and on the intracellular free calcium concentration ([Ca2+]i) in cultured endothelial cells from porcine aorta. In patch-clamped cells, pinacidil (1 mumol/l) and cromakalim (1 mumol/l) elicited outward currents carried by K+ and significantly prolonged the Ca2(+)-dependent K+ currents induced by bradykinin and ATP. Peak currents in response to bradykinin were not affected. In cells loaded with the fluorescent Ca2+ indicator indo-1 and prestimulated with thimerosal, pinacidil (0.1-1 mumol/l elicited long-lasting increases in [Ca2+]i from 100 +/- 10 to 550 +/- 110 nmol/l. These effects were completely abolished in a medium containing 90 mmol/l K+. Similar results were obtained with cromakalim. Likewise, in cells stimulated with bradykinin, pinacidil raised [Ca2+]i when applied during the decline of [Ca2+]i after the initial peak. Cicletanine elicited K+ currents in resting and attenuated K+ currents in bradykinin-stimulated cells. It elevated [Ca2+]i even in the absence of extracellular Ca2+ and in K(+)-rich medium. Hence, the effects of cicletanine cannot be explained by direct actions on K+ channels. However, our studies demonstrate that pinacidil and cromakalim elevate [Ca2+]i secondary to their activation of K+ channels by inducing hyperpolarization and augmenting the driving force for potential-dependent Ca2+ influx. In this way, the two drugs may promote Ca2(+)-dependent formation of endothelium-derived relaxing factor.

The synthetic lipopeptide Pam3Cys-Ala-Gly, an analogue of the N-terminal part of bacterial lipoprotein, constitutes a potent macrophage activator. The role of protein kinase C (PKC) in lipopeptide induced signal transduction was investigated. As determined by enzymatic and immunochemical methods, translocation of PKC could not be observed in lipopeptide stimulated bone marrow derived macrophages. Our studies showed that the membrane-associated form of PKC displayed different characteristics than the cytosolic form. The second messengers, inositoltrisphosphate, cAMP and cGMP, did not seem to be involved in signal transduction. Unlike LPS, Pam3Cys-Ala-Gly induced a rapid rise in cytosolic Ca2+, which was due to an influx of extracellular calcium as well as to a redistribution of intracellular calcium. The data suggest that one major intracellular signal transduction mechanism initiated by lipopeptide consists of altering internal Ca2+ concns.

The release of vasodilating substances from the vascular endothelium has been postulated to depend on a rise in the level of intracellular free calcium (Cai++). We measured Cai++ in intact monolayers of calf endothelial cells, grown in culture, that were loaded with the fluorescent calcium indicator quin 2. Fluorescence (excitation wavelength 340 nm, emission wavelength 492 nm) was calibrated by raising Cai++ to a maximum with the calcium ionophore ionomycin (0.1 microM) and by lowering it to a minimum with ionomycin plus manganese (0.4 mM), which quenches quin 2 fluorescence completely. Loss of fluorescent dye from the cells was calculated from fluorescence at the isosbestic excitation wavelength (365 nm). Resting Cai++ was 71 +/- 3 (SEM) nM. ATP (adenosine-5'-triphosphate) raised Cai++ dose-dependently and reversibly to 458 +/- 60 nM at a concentration of 10 microM, and at 0.1 mM to values close to those that occurred under ionomycin. ADP (A-5'-PP) and AMP (A-5'-P) had smaller effects with a maximal Cai++ of 287 +/- 72 nM at 30 microM ADP and 176 +/- 17 nM at 0.1 mM AMP. At these concentrations, ADP and AMP attenuated significantly the increase of Cai++ under ATP (10 microM). Adenosine (0.1 or 0.3 mM) and acetylcholine (0.1 to 30 microM) enhanced Cai++ inconsistently, by a maximum of 50 nM. These effects were abolished by theophylline and atropine, respectively. In the absence of extracellular calcium, ATP still raised Cai++, although endothelial responsiveness declined after repetitive stimulations. We conclude that activation of purinergic receptors increases intracellular free calcium in endothelial cells, and that this increase is probably an essential trigger for synthesis of prostacyclin and the labile endothelium-derived relaxant factor.

In this article we compare electrical conductance events from single channel recordings of three TRP channel proteins (TRPA1, TRPM2 and TRPM8) expressed in human embryonic kidney cells with channel events recorded on synthetic lipid membranes close to melting transitions. Ion channels from the TRP family are involved in a variety of sensory processes including thermo- and mechano-reception. Synthetic lipid membranes close to phase transitions display channel-like events that respond to stimuli related to changes in intensive thermodynamic variables such as pressure and temperature. TRP channel activity is characterized by typical patterns of current events dependent on the type of protein expressed. Synthetic lipid bilayers show a wide spectrum of electrical phenomena that are considered typical for the activity of protein ion channels. We find unitary currents, burst behavior, flickering, multistep-conductances, and spikes behavior in both preparations. Moreover, we report conductances and lifetimes for lipid channels as described for protein channels. Non-linear and asymmetric current-voltage relationships are seen in both systems. Without further knowledge of the recording conditions, no easy decision can be made whether short current traces originate from a channel protein or from a pure lipid membrane, Comment: 13 pages, 9 Figures

Depletion of intracellular calcium stores induces transmembrane Ca2+ influx. We studied Ca(2+)- and Ba(2+)-permeable ion channels in A431 cells after store depletion by dialysis of the cytosol with 10 mM BAPTA solution. Cell-attached patches of cells held at low (0.5 microM) external Ca2+ exhibited transient channel activity, lasting for 1-2 min. The channel had a slope conductance of 2 pS with 200 mM CaCl2 and 16 pS with 160 mM BaCl2 in the pipette. Channel activity quickly ran down in excised inside-out patches and was not restored by InsP3 and/or InsP4. Thapsigargin induced activation in cells kept in 1 mM external Ca2+ after BAPTA dialysis. These channels represent one Ca2+ entry pathway activated by depletion of internal calcium stores and are clearly distinct from previously identified calcium repletion currents.

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca2+ influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca2+ influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

We studied the effects and the mechanism of action of the cyclic GMP-lowering substance 6-anilino-5,8-quinolinedione (LY 83583) on cyclic GMP-mediated inhibition of platelet function. The activation of washed human platelets by thrombin was counteracted by 8-bromo-cyclic GMP and the direct activators of soluble guanylate cyclase, sodium nitroprusside and endothelium-derived relaxant factor (EDRF = nitric oxide). LY 83583 significantly antagonized the inhibitory effect of sodium nitroprusside and EDRF, but not that of 8-bromo-cyclic GMP, on thrombin-induced aggregation, ATP-release, adhesion to native endothelial cells and increase in concentration of free intracellular calcium ions. In accordance, increases in intracellular cyclic GMP by sodium nitroprusside and EDRF were attenuated by LY 83583. The inhibition of cyclic GMP-mediated effects on platelets by LY 83583 could be related to inhibition of platelet soluble guanylate cyclase, as the activation of the purified enzyme from platelets by sodium nitroprusside was directly inhibited by LY 83583. This effect of LY 83583 was attenuated in the presence of superoxide dismutase. Our findings support the hypothesis that sodium nitroprusside and EDRF inhibit platelet activation by stimulation of soluble guanylate cyclase via nitric oxide. Consequently, inhibition of nitric oxide-induced cyclic GMP formation by LY 83583, which may act by intracellular generation of superoxide anions, facilitates platelet activation.

The distal colon (rat) was perfused in vivo at low rates (1-2 ml/h). Dialyzed polyethyleneglycol 4,000 (PEG) was used to vary the luminal osmotic activity (pi eff). Perfusate sodium concentration of 3.7 mmol 1-1 +/- 0.9 (SD) resulted in an effluent Na+ of 3.3 mmol 1-1 +/- 1.4. Potassium concentrations of 15 or 25 mmol 1-1 remained unaltered. At this cationic steady-state, transmural net water flux (Jv) was linearly correlated (r = -0.96, n = 24) to the logarithm of pi eff. Jv was zero at 634 mosm 1-1. Lp (microliters h-1 cm-2 mosm-1 l) was 0.195 at pi eff 85 mosm 1-1 and 0.046 at 1,050 mosm 1-1. These data characterize the distal colon as an epithelium with high capacity for salt conservation.