Greg J. Barritt

RECEPTOR-ACTIVATED CA2+ INFLOW IN ANIMAL CELLS : A VARIETY OF PATHWAYS TAILORED TO MEET DIFFERENT INTRACELLULAR CA2+ SIGNALLING REQUIREMENTS

Receptor-activated Ca2+ channels (RACCs) play a central role in regulation of the functions of animal cells. Together with voltage-operated Ca2+ channels (VOCCs) and ligand-gated non-selective cation channels, RACCs provide a variety of pathways by which Ca2+ can be delivered to the cytoplasmic space and the endoplasmic reticulum (ER) in order to initiate or maintain specific types of intracellular Ca2+ signal. Store-operated Ca2+ channels (SOCs), which are activated by a decrease in Ca2+ in the ER, are a major subfamily of RACCs. A careful analysis of the available data is required in order to discern the different types of RACCs (differentiated chiefly on the basis of ion selectivity and mechanism of activation) and to properly develop hypotheses for structures and mechanisms of activation. Despite much intensive research, the structures and mechanisms of activation of RACCs are only now beginning to be understood. In considering the physiological functions of the different RACCs, it is useful to consider the specificity for Ca2+ of each type of cation channel and the rate at which Ca2+ flows through a single open channel; the locations of the channels on the plasma membrane (in relation to the ER, cytoskeleton and other intracellular units of structure and function); the Ca2+-responsive enzymes and proteins; and the intracellular buffers and proteins that control the distribution of Ca2+ in the cytoplasmic space. RACCs which are non-selective cation channels can deliver Ca2+ directly to specific regions of the cytoplasmic space, and can also admit Na+, which induces depolarization of the plasma membrane, the opening of VOCCs and the subsequent inflow of Ca2+. SOCs appear to deliver Ca2+ specifically to the ER, thereby maintaining oscillating Ca2+ signals.

Evidence that 2-aminoethyl diphenylborate is a novel inhibitor of store-operated Ca2+ channels in liver cells, and acts through a mechanism which does not involve inositol trisphosphate receptors.

The compound 2-aminoethyl diphenylborate (2-APB), an inhibitor of Ins(1,4,5)P(3) receptor action in some cell types, has been used to assess the role of Ins(1,4,5)P(3) receptors in the activation of store-operated Ca2+ channels (SOCs) [Ma, Patterson, van Rossum, Birnbaumer, Mikoshiba and Gill (2000) Science 287, 1647-1651]. In freshly-isolated rat hepatocytes, 2-APB inhibited thapsigargin- and vasopressin-stimulated Ca2+ inflow (measured using fura-2) with no detectable effect on the release of Ca2+ from intracellular stores. The concentration of 2-APB which gave half-maximal inhibition of Ca2+ inflow was approx. 10 microM. 2-APB also inhibited Ca2+ inflow initiated by a low concentration of adenophostin A but had no effect on maitotoxin-stimulated Ca2+ inflow through non-selective cation channels. The onset of the inhibitory effect of 2-APB on thapsigargin-stimulated Ca2+ inflow was rapid. When 2-APB was added to rat hepatocytes in the presence of extracellular Ca2+ after a vasopressin-induced plateau in the cytoplasmic free Ca2+ concentration ([Ca2+](cyt)) had been established, the kinetics of the decrease in [Ca2+](cyt) were identical with those induced by the addition of 50 microM Gd(3+) (gadolinium). 2-APB did not inhibit the release of Ca2+ from intracellular stores induced by the addition of Ins(1,4,5)P(3) to permeabilized hepatocytes. In the H4-IIE rat hepatoma cell line, 2-APB inhibited thapsigargin-stimulated Ca2+ inflow (measured using fura-2) and, in whole-cell patch-clamp experiments, the Ins(1,4,5)P(3)-induced inward current carried by Ca2+. It was concluded that, in liver cells, 2-APB inhibited SOCs through a mechanism which involved the binding of 2-APB to either the channel protein or an associated regulatory protein. 2-APB appeared to be a novel inhibitor of SOCs in liver cells with a mechanism of action which, in this cell type, is unlikely to involve an interaction of 2-APB with Ins(1,4,5)P(3) receptors. The need for caution in the use of 2-APB as a probe for the involvement of Ins(1,4,5)P(3) receptors in the activation of SOCs in other cell types is briefly discussed.

Evidence that TRPC1 (transient receptor potential canonical 1) forms a Ca2+-permeable channel linked to the regulation of cell volume in liver cells obtained using small interfering RNA targeted against TRPC1

The TRPC1 (transient receptor potential canonical 1) protein, which is thought to encode a non-selective cation channel activated by store depletion and/or an intracellular messenger, is expressed in a number of non-excitable cells. However, the physiological functions of TRPC1 are not well understood. The aim of these studies was to investigate the function of TRPC1 in liver cells using small interfering RNA (siRNA) to ablate the TRPC1 protein. Treatment of H4-IIE liver cells with siRNA targeted against TRPC1 caused an approx. 50% decrease in expression of the human TRPC1 protein in cells transfected with cDNA encoding human TRPC1, and a 50% decrease in expression of the endogenous TRPC1 protein (assessed by Western blot and immunofluorescence). The decrease in endogenous TRPC1 protein in cells transfected with TRPC1 siRNA was associated with a greater increase in cell volume (compared with the increase observed in control cells) immediately after cells were placed in a hypotonic medium, and an enhanced regulatory cell volume decrease after exposure to hypotonic medium. Treatment with siRNA targeted against TRPC1 also led to a 25% inhibition of thapsigargin-stimulated Ca(2+) inflow, a 40% inhibition of ATP and maitotoxin-stimulated Ca(2+) inflow, and a 50% inhibition of maitotoxin-stimulated Mn(2+) inflow. The idea that, in liver cells, TRPC1 encodes a non-selective cation channel involved directly or indirectly in the regulation of cell volume is consistent with the results obtained.

Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis

Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80 kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92 kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the alpha- and beta-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92 kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120 kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92 kDa or 120 kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated beta-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.

TRPM2 channels mediate acetaminophen-induced liver damage

Significance Acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. There are limited options for early treatment. Acetaminophen liver toxicity leads to the formation of reactive oxygen and nitrogen species which cause an increase in intracellular Ca2+ and hepatocellular death. We show that acetaminophen-induced liver toxicity depends on Transient Receptor Potential Melanostatine 2 (TRPM2) cation channels in hepatocytes, which are activated in response to oxidative stress and are responsible for Ca2+ overload. Lack of TRPM2 channels in hepatocytes or their pharmacological inhibition protects liver from acetaminophen toxicity. This provides evidence that TRPM2 may present a potential therapeutic target for treatment of oxidative-stress related liver diseases. Acetaminophen (paracetamol) is the most frequently used analgesic and antipyretic drug available over the counter. At the same time, acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. Acetaminophen overdose causes a multitude of interrelated biochemical reactions in hepatocytes including the formation of reactive oxygen species, deregulation of Ca2+ homeostasis, covalent modification and oxidation of proteins, lipid peroxidation, and DNA fragmentation. Although an increase in intracellular Ca2+ concentration in hepatocytes is a known consequence of acetaminophen overdose, its importance in acetaminophen-induced liver toxicity is not well understood, primarily due to lack of knowledge about the source of the Ca2+ rise. Here we report that the channel responsible for Ca2+ entry in hepatocytes in acetaminophen overdose is the Transient Receptor Potential Melanostatine 2 (TRPM2) cation channel. We show by whole-cell patch clamping that treatment of hepatocytes with acetaminophen results in activation of a cation current similar to that activated by H2O2 or the intracellular application of ADP ribose. siRNA-mediated knockdown of TRPM2 in hepatocytes inhibits activation of the current by either acetaminophen or H2O2. In TRPM2 knockout mice, acetaminophen-induced liver damage, assessed by the blood concentration of liver enzymes and liver histology, is significantly diminished compared with wild-type mice. The presented data strongly suggest that TRPM2 channels are essential in the mechanism of acetaminophen-induced hepatocellular death.

Evidence for the expression of transient receptor potential proteins in guinea pig airway smooth muscle cells.

Objective:  The present study investigates the expression of transient receptor potential (TRPC) proteins in airway smooth muscle (ASM) cells in order to determine whether these proteins may be candidate molecular counterparts of plasma membrane Ca2+‐permeable channels involved in the contraction of ASM.

Novel variants of voltage-operated calcium channel α1-subunit transcripts in a rat liver-derived cell line: deletion in the IVS4 voltage sensing region

Using reverse transcriptase-PCR and Northern analysis, we have shown that the H4IIE cell line, derived from the Reuber H35 rat hepatoma, contains significant amounts of transcripts for the CaCh3 (neuroendocrine) and CaCh1 (skeletal muscle) L-type voltage-operated calcium channel alpha 1-subunits. Two of the CaCh3 transcripts have a 45 bp deletion in the IVS4 membrane-spanning region which is the result of a mutation in genomic DNA. The deduced amino acid sequences of the PCR-derived clones of CaCh3 indicate that the mutation causes the loss of 15 amino acids from the IVS4 region, including three of the six positively charged residues, which are thought to be part of the voltage-sensing mechanism of voltage-operated Ca2+ channels. Quantitative-PCR and Northern analysis indicate that one of the novel CaCh3 transcripts is present in sufficient amounts to imply it could play a functional role in Ca2+ inflow. RT-PCR analysis of hepatocytes isolated from rat liver detected transcripts of CaCh3 (without the IVS4 mutation) and CaCh2, but at considerably lower levels than observed for the isoforms in the H4IIE cell line. Transcripts of CaCh1 and CaCh2 were also detected at low levels in Jurkat T lymphocytes. Fluorimetric studies with the Ca(2+)-sensitive probe, Fluo-3, have shown that H4IIE cells exhibit receptor-activated and store-activated (thapsigarin-induced), but not depolarisation (extracellular KCl)-induced Ca2+ inflow. The mutant transcripts are unlikely to produce Ca2+ channels that are opened by membrane depolarisation. The idea that they may be opened by other mechanisms is briefly discussed.

Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes.

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca2+ channel, in store-operated Ca2+ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3′ and 5′ rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-1 polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca2+ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP3F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC50 = 0.5 μM), indicating that InsP3F and LPA principally activate store-operated Ca2+ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP3F-, LPA- or thapsigargin-stimulated Ca2+ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP3F-stimulated Ca2+ inflow and only a very small enhancement of LPA-stimulated Ca2+ inflow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca2+ inflow through the previously-characterised Ca2+-specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.

Phospholipase C-γ1 is required for the activation of store-operated Ca2+ channels in liver cells

Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-gamma (phospholipase C-gamma) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-gamma-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-gamma has not been defined clearly. The aim of the present study was to determine if PLC-gamma is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-gamma1 caused a reduction (by approx. 70%) in the PLC-gamma1 protein expression, with maximal effect at 72-96 h. This was associated with a decrease (by approx. 60%) in the amplitude of the I(SOC) (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP(3) or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the I(SOC) amplitude. Immunoprecipitation of PLC-gamma1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-gamma1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.

Characterisation of the divalent cation channels of the hepatocyte plasma membrane receptor-activated Ca2+ inflow system using lanthanide ions.

The ability of Gd3+ to inhibit vasopressin-stimulated Ca2+ inflow to hepatocytes was compared with its effect on Mn2+ inflow. In the absence of Gd3+, the stimulation of Mn2+ inflow by vasopressin increased with increasing pH of the extracellular medium. Maximal inhibition of vasopressin-stimulated Ca2+ and Mn2+ inflow by saturating concentrations of Gd3+ was 70 and 30%, respectively. Gd3+ also inhibited thapsigargin-stimulated Ca2+ and Mn2+ inflow with maximal inhibition of 70 and 40%, respectively. It is concluded that vasopressin and thapsigargin each activate two types of Ca2+ inflow processes, one which is sensitive and one which is insensitive to lanthanides. The nature of the pore of the lanthanide-sensitive Ca2+ channel was investigated further using different lanthanides as inhibitors. Tm3+, Gd3+, Eu3+, Nd3+ and La3+ each inhibited vasopressin-stimulated Ca2+ and Mn2+ inflow but had no effect on Ca2+ inflow in the absence of an agonist, or on vasopressin-stimulated release of Ca2+ from intracellular stores. Maximal inhibition of vasopressin-stimulated Ca2+ inflow in the presence of a saturating concentration of each lanthanide ranged from 70-90%. An equation which describes a 1:1 interaction of the lanthanide with a putative binding site in the Ca2+ channel gave a good fit to dose-response curves for the inhibition of vasopressin-stimulated Ca2+ inflow by each lanthanide. Lanthanides in the middle of the series exhibited the lowest dissociation constant (Kd) values. The Kd for Gd3+ increased with increasing extracellular Ca2+ concentration, suggesting competitive inhibition of Ca2+ binding by Gd3+. In the absence of lanthanide, vasopressin-stimulated Mn2+ inflow was substantially reduced when the plasma membrane was depolarised by increasing the extracellular K+ concentration. Changing the membrane potential had little effect on the maximum inhibition by Gd3+ of vasopressin-stimulated Mn2+ inflow. The Kd for inhibition of vasopressin-stimulated Ca2+ inflow by Gd3+, measured at the lowest attainable membrane potential, was about 6-fold lower than the Kd measured at the highest attainable membrane potential. The idea that there is a site in the vasopressin-stimulated lanthanide-sensitive Ca2+ channel composed of carboxylic acid groups which bind Ca2+, Mn2+ or a lanthanide ion is consistent with the data obtained using the different lanthanides.