Andreas Lückhoff

Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD.

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.

The TRP family of cation channels: probing and advancing the concepts on receptor-activated calcium entry

Stimulation of membrane receptors linked to a phospholipase C and the subsequent production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (InsP(3)) is a signaling pathway of fundamental importance in eukaryotic cells. Signaling downstream of these initial steps involves mobilization of Ca(2+) from intracellular stores and Ca(2+) influx through the plasma membrane. For this influx, several contrasting mechanisms may be responsible but particular relevance is attributed to the induction of Ca(2+) influx as consequence of depletion of intracellular calcium stores. This phenomenon (frequently named store-operated calcium entry, SOCE), in turn, may be brought about by various signals, including soluble cytosolic factors, interaction of proteins of the endoplasmic reticulum with ion channels in the plasma membrane, and a secretion-like coupling involving translocation of channels to the plasma membrane. Experimental approaches to analyze these mechanisms have been considerably advanced by the discovery of mammalian homologs of the Drosophila cation channel transient receptor potential (TRP). Some members of the TRP family can be expressed to Ca(2+)-permeable channels that enable SOCE; other members form channels activated independently of stores. TRP proteins may be an essential part of endogenous Ca(2+) entry channels but so far expression of most TRP cDNAs has not resulted in restitution of channels found in any mammalian cells, suggesting the requirement for further unknown subunits. A major exception is CaT1, a TRP channel demonstrated to provide Ca(2+)-selective, store-operated currents identical to those characterized in several cell types. Ongoing and future research on TRP channels will be crucial to understand the molecular basis of receptor-mediated Ca(2+) entry, with respect to the structure of the entry channels as well as to the mechanisms of its activation and regulation.

Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3.

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.

TRPM2: a calcium influx pathway regulated by oxidative stress and the novel second messenger ADP-ribose

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 Ca2+ enhances TRPM2 gating by ADPR; ADPR and Ca2+ in concert may be an important messenger system mediating Ca2+ influx. Other stimuli of TRPM2 include NAD and H2O2 and cyclic ADPR, which may act synergistically with ADPR. H2O2, 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.

Effects of the Anesthetic Gases Xenon, Halothane, and Isoflurane on Calcium and Potassium Currents in Human Atrial Cardiomyocytes

BackgroundNegative inotropic and proarrhythmic side effects on the heart are well known for the volatile anesthetics halothane and isoflurane but not for the noble gas xenon. We investigated the effects of halothane, isoflurane, and xenon on calcium and potassium currents in human atrial myocytes to elucidate the cellular and molecular basis of their cardiac actions. MethodsAtrial myocytes were prepared from the right auricles obtained from patients undergoing heart surgery. Ion currents were measured with the whole cell patch clamp technique during superfusion of the cells with solutions that contained halothane, isoflurane, or xenon at concentrations corresponding to their respective minimum alveolar concentration (MAC); gas concentrations were determined with the head space–gas chromatography/mass spectrometry/selected ion monitoring method. ResultsL-type calcium currents were significantly depressed by 31.9 ± 4.1%, from −1.8 ± 0.3 to −1.2 ± 0.4 picoampere (pA)/picofarad (pF) (n = 4;P < 0.05) at 1 MAC halothane and by 21.7 ± 9.2%, from −1.6 ± 0.7 to −1.2 ± 0.6 pA/pF (n = 7;P < 0.05) at 1 MAC isoflurane, but not affected by 70% xenon (1 MAC). Inwardly rectifying potassium currents were not influenced by any anesthetic. Halothane (1 MAC) significantly inhibited the transient as well as the sustained part of voltage-gated potassium outward currents, by 19.4 ± 6.7%, from 6.7 ± 2.1 to 5.4 ± 1.6 pA/pF (n = 8;P < 0.05), and by 8.6 ± 4.8%, from 5.5 ± 1.7 to 5.0 ± 1.5 pA/pF (n = 8;P < 0.05), respectively. Transient K+ outward currents were even more inhibited, by 25.8 ± 4.8%, from 9.8 ± 3.1 to 7.3 ± 2.1 pA/pF (n = 5;P < 0.05) at 1 MAC isoflurane, whereas xenon evoked only a slight (albeit significant) inhibition, by 6.1 ± 3.7%, from 8.2 ± 6.0 to 7.7 ± 5.8 pA/pF (n = 10;P < 0.05). Isoflurane and xenon did not affect sustained potassium currents. All effects of the anesthetics were fully reversible after washout. ConclusionsHalothane and isoflurane exhibited considerable inhibitory effects on voltage-gated cardiac Ca2+ and K+ currents important for the duration of action potentials and the repolarization. Xenon, in contrast, did not affect Ca2+ currents and only slightly inhibited transient K+ outward currents, in line with the almost absent cardiac side effects of the noble gas.

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1β

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 TRP1β (mTRP1β) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1β expressing cells exhibited enhanced Ca2+ influx and enhanced whole‐cell membrane currents compared to mTRP1β 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 mTRP1β is required for structural organization thus forming functional channels.

H2O2-induced Ca2+ influx and its inhibition by N-(p-amylcinnamoyl) anthranilic acid in the β-cells: involvement of TRPM2 channels

Type 2 melastatin‐related transient receptor potential channel (TRPM2), a member of the melastatin‐related TRP (transient receptor potential) subfamily is a Ca2+‐permeable channel activated by hydrogen peroxide (H2O2). We have investigated the role of TRPM2 channels in mediating the H2O2‐induced increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in insulin‐secreting cells. In fura‐2 loaded INS‐1E cells, a widely used model of β‐cells, and in human β‐cells, H2O2 increased [Ca2+]i, in the presence of 3 mM glucose, by inducing Ca2+ influx across the plasma membrane. H2O2‐induced Ca2+ influx was not blocked by nimodipine, a blocker of the L‐type voltage‐gated Ca2+ 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 H2O2, 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 H2O2‐induced Ca2+ entry in β‐cells, a process potently inhibited by ACA.

Impairment of glucose metabolism in hearts from rats treated with endotoxin

OBJECTIVE In patients and animals with sepsis or critical illness, the mechanical function of the heart is often impaired. Although these conditions are accompanied by dramatic metabolic and hormonal changes, little is known about alterations of cardiac metabolism. In this study, we assessed the impact of an endotoxin-induced inflammation on cardiac glucose utilization. METHODS Bacterial endotoxin (1 mg/kg lipopolysaccharide from Salmonella typhimurium, LPS) was injected intravenously to rats. Six hours after LPS application, hearts were isolated and perfused in the Langendorff mode. RESULTS Left ventricular pressure was reduced by 50% in hearts from LPS-treated rats, compared to those from saline-injected control animals. With glucose as the sole fuel, there was no difference in glycolysis between the groups. However, on addition of beta-hydroxybutyrate (an alternative fuel which inhibits phosphofructokinase via an increased citrate level), the glycolytic rate in the LPS group was 44 and 48% lower (in basal, and insulin-stimulated conditions, respectively; P<0.01) than in control hearts. At the end of perfusions with beta-hydroxybutyrate and insulin, the cardiac citrate content was 40% higher in LPS vs. controls (P<0.001). In addition to the reduced glycolysis, the insulin-dependent increase of cardiac glycogen was 77% smaller in LPS hearts. The difference between LPS and control glycolysis was abolished if the hearts were perfused with the ceramidase inhibitor N-oleyl-ethanolamine (5 microM), and also with the cyclooxygenase-2 inhibitor NS-398 (10 microM), or the thromboxane A2 receptor antagonist SQ-29548 (1 microM). CONCLUSION The inflammatory reaction caused by endotoxin impairs cardiac glucose metabolism (and in particular, the action of insulin) in at least two ways: through the exacerbation of the counterregulatory effect of alternative fuels on glycolysis, and through a reduction in net glycogen synthesis. Impairment of glycolysis may be mediated by a sphingomyelin derivative, and COX-2-derived thromboxane A2.

Regulation of TRPM2 channels in neutrophil granulocytes by ADP-ribose: a promising pharmacological target

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 Ca2+, 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 Ca2+ ions from intracellular stores which in cooperation with endogenous ADPR levels enable Ca2+ 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.

Inhibition of TRPM8 by Icilin Distinct from Desensitization Induced by Menthol and Menthol Derivatives

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 Ca2+-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 Ca2+-independent manner as well as inducing Ca2+-dependent desensitization. Icilin, in contrast, activated currents only in wild type TRPM8 and in the presence of Ca2+. 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 Ca2+. 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.