[Ca2+ influx mechanisms activated by receptors: capacitative Ca2+ entry and TRP proteins].
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Amino Acid Sequence↗
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A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade.
How are signalling molecules organized into different pathways within the same cell? In Drosophila, the inaD gene encodes a protein consisting of five PDZ domains which serves as a scaffold to assemble different components of the phototransduction cascade, including the principal light-activated ion channels, the effector phospholipase C-beta and protein kinase C. Null inaD mutants have a dramatically reorganized subcellular distribution of signalling molecules, and a total loss of transduction complexes. Also, mutants defective in a single PDZ domain produce signalling complexes that lack the target protein and display corresponding defects in their physiology. A picture emerges of a highly organized unit of signalling, a 'transduclisome', with PDZ domains functioning as key elements in the organization of transduction complexes in vivo.
Amino Acid Sequence↗
Light-dependent subcellular translocation of Gqalpha in Drosophila photoreceptors is facilitated by the photoreceptor-specific myosin III NINAC.
We examine the light-dependent subcellular translocation of the visual G(q)alpha protein between the signaling compartment, the rhabdomere and the cell body in Drosophila photoreceptors. We characterize the translocation of G(q)alpha and provide the first evidence implicating the involvement of the photoreceptor-specific myosin III NINAC in G(q)alpha transport. Translocation of G(q)alpha from the rhabdomere to the cell body is rapid, taking less than 5 minutes. Higher light intensities increased the quantity of G(q)alpha translocated out of the rhabdomeres from 20% to 75%, consistent with a mechanism for light adaptation. We demonstrate that translocation of G(q)alpha requires rhodopsin, but none of the known downstream phototransduction components, suggesting that the signaling pathway triggering translocation occurs upstream of G(q)alpha. Finally, we show that ninaC mutants display a significantly reduced rate of G(q)alpha transport from the cell body to the rhabdomere, suggesting that NINAC might function as a light-dependent plus-end motor involved in the transport of G(q)alpha.
Animals↗
Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor.
Brain-derived neurotrophic factor (BDNF) is known to promote neuronal survival and differentiation and to guide axon extension both in vitro and in vivo. The BDNF-induced chemo-attraction of axonal growth cones requires Ca2+ signalling, but how Ca2+ is regulated by BDNF at the growth cone remains largely unclear. Extracellular application of BDNF triggers membrane currents resembling those through TRPC (transient receptor potential canonical) channels in rat pontine neurons and in Xenopus spinal neurons. Here, we report that in cultured cerebellar granule cells, TRPC channels contribute to the BDNF-induced elevation of Ca2+ at the growth cone and are required for BDNF-induced chemo-attractive turning. Several members of the TRPC family are highly expressed in these neurons, and both Ca2+ elevation and growth-cone turning induced by BDNF are abolished by pharmacological inhibition of TRPC channels, overexpression of a dominant-negative form of TRPC3 or TRPC6, or downregulation of TRPC3 expression via short interfering RNA. Thus, TRPC channel activity is essential for nerve-growth-cone guidance by BDNF.
Animals↗
Isolation of genes encoding photoreceptor-specific proteins by immunoscreening with antibodies directed against purified blowfly rhabdoms.
The proteins which perform and regulate key steps in phototransduction are assumed to be localized in the rhabdomeric membrane of invertebrate photoreceptor cells. We have employed antibodies raised against rhabdoms purified from blowfly eyes in order to isolate copy deoxyribonucleic acid (cDNA) clones encoding proteins that are required in the phototransduction machinery. By immunoscreening a Calliphora retinal cDNA library, we obtained clones of genes coding for five different proteins. As revealed by partial cDNA sequence analysis, three of these genes represent the Calliphora homologs of Drosophila trp, inaC and InaD, while the other two displayed no homology to known genes. Northern blot analysis confirmed that trp, inaC and InaD transcripts were present in RNA isolated from the retina, but not in RNA isolated from brain or thorax. Specific antibodies directed against trp, inaC and InaD protein were raised using recombinantly expressed proteins or synthetic peptides. Western blot analyses revealed that trp, inaC and InaD protein are specifically associated with the rhabdomeral photoreceptor membrane. Extraction of membranes with buffers of different ionic strengths suggested that the trp gene product is an integral membrane protein, whilst the inaC and InaD gene products are peripherally bound membrane proteins. This demonstrates that the immunoscreening approach used here can be successfully applied to isolate genes that code for either integral or peripheral photoreceptor membrane proteins.
Amino Acid Sequence↗
ATP-induced mitogenesis is mediated by cyclic AMP response element-binding protein-enhanced TRPC4 expression and activity in human pulmonary artery smooth muscle cells.
Extracellular ATP and intracellular cyclic AMP response element-binding protein (CREB, a transcription factor) promote cell proliferation in many cell types. The canonical transient receptor potential (TRPC) channels, which putatively participate in forming store- and receptor-operated Ca2+ channels, have been implicated in the pulmonary vascular remodeling processes. A link between extracellular ATP, CREB activation, and TRPC4 channel expression and activity has not been shown in human pulmonary artery smooth muscle cells (PASMC). Long-term (24-48 h) treatment of human PASMC with a low dose (100 microM) of ATP, which did not trigger a transient rise in free cytosolic Ca2+ concentration ([Ca2+]i) when applied acutely to the cells, caused marked increases in CREB phosphorylation and TRPC4 protein expression. The time course indicated that the ATP-mediated CREB phosphorylation preceded TRPC4 upregulation, whereas transfection of a nonphosphorylatable CREB mutant abolished ATP-mediated TRPC4 expression. Furthermore, treatment of human PASMC with ATP also enhanced the amplitude of capacitative Ca2+ entry (CCE) induced by passive store depletion, whereas the small interfering RNA specifically targeting TRPC4 attenuated ATP-mediated increases in TRPC4 expression and CCE amplitude and inhibited ATP-induced PASMC proliferation. These data suggest that low-dose ATP exerts part of its mitogenic effect in human PASMC via CREB-mediated upregulation of TRPC4 channel expression and activity and the subsequent increase in CCE and [Ca2+]i.
Adenosine Triphosphate↗
The relationship of TRP channels to the pacemaker activity of interstitial cells of Cajal in the gastrointestinal tract.
Interstitial cells of Cajal (ICCs) are a fundamental component of the pacemaker apparatus of the gastrointestinal (GI) tract. They have special properties that make them unique in their ability to generate and propagate slow waves in gastrointestinal smooth muscle. The pacemaker current that generates slow waves is initially due to a voltage-independent, Ca(2+)-inhibited, non-selective cationic conductance in ICC. The classical transient receptor potential (TRPC) channel 4 was suggested as a molecular candidate for the nonselective cation channel (NSCC) responsible for the pacemaker activity. We have shown that TRPC4-/- mice display normal slow waves and suggest that TRPC4 might be an essential component of the NSCC activated by muscarinic stimulation. Finally, we suggest that TRPM7 is the molecular candidate for the NSCC responsible for pacemaker activity in ICCs on the basis of electrophysiological, molecular biological, and immunohistochemical experiments.
Animals↗
PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression.
Capacitative Ca(2+) entry (CCE) through store-operated Ca(2+) (SOC) channels plays an important role in returning Ca(2+) to the sarcoplasmic reticulum (SR) and regulating cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)). A rise in [Ca(2+)](cyt) and sufficient Ca(2+) in the SR are required for pulmonary artery smooth muscle cell (PASMC) proliferation. We tested the hypothesis that platelet-derived growth factor (PDGF)-mediated PASMC growth involves upregulation of c-Jun and TRPC6, a transient receptor potential cation channel. In rat PASMC, PDGF (10 ng/ml for 0.5-48 h) phosphorylated signal transducer and activator of transcription (STAT3), increased mRNA and protein levels of c-Jun, and stimulated cell proliferation. PDGF treatment also upregulated TRPC6 expression and augmented CCE, elicited by passive depletion of Ca(2+) from the SR using cyclopiazonic acid. Furthermore, overexpression of c-Jun stimulated TRPC6 expression and CCE amplitude in PASMC. Downregulation of TRPC6 using an antisense oligonucleotide specifically for human TRPC6 decreased CCE and inhibited PDGF-mediated PASMC proliferation. These results suggest that PDGF-mediated PASMC proliferation is associated with c-Jun/STAT3-induced upregulation of TRPC6 expression. The resultant increase in CCE raises [Ca(2+)](cyt), facilitates return of Ca(2+) to the SR, and enhances PASMC growth.
Animals↗
Receptor-induced activation of Drosophila TRP gamma by polyunsaturated fatty acids.
Cellular calcium homeostasis is regulated by hormones and neurotransmitters, resulting in the activation of a variety of proteins, in particular, channel proteins of the plasma membrane and of intracellular compartments. Such channels are, for example, TRP channels of the TRPC protein family that are activated by various mediators from receptor-stimulated signaling cascades. In Drosophila, two TRPC channels, TRP and TRPL, are involved in phototransduction. In addition, a third Drosophila TRPC channel, TRPgamma, has been identified and described as an auxiliary subunit of TRPL. Beyond it, our data show that heterologously expressed TRPgamma formed a receptor-activated, outwardly rectifying cation channel independent from TRPL co-expression. Analysis of the activation mechanism revealed that TRPgamma is activated by various polyunsaturated fatty acids generated in a phospholipase C- and phospholipase A(2)-dependent manner. The most potent activator of TRPgamma, the stable analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid, induced currents in single channel recordings. Here we show that upon heterologous expression TRPgamma forms a homomeric channel complex that is activated by polyunsaturated fatty acids as mediators of receptor-dependent signaling pathways. Reverse transcription PCR analysis showed that TRPgamma is expressed in Drosophila heads and bodies. Its body-wide expression pattern and its activation mechanism suggest that TRPgamma forms a fly cation channel responsible for the regulation of intracellular calcium in a variety of hormonal signaling cascades.
Animals↗
The COOH-terminal domain of Drosophila TRP channels confers thapsigargin sensitivity.
Previous studies have shown that the Drosophila cation channels designated Trp and Trpl can be functionally expressed in Sf9 insect cells using baculovirus expression vectors. The trp gene encodes a Ca2+-permeable channel that is activated by thapsigargin, blocked by low micromolar Gd3+, and is relatively selective for Ca2+ versus Na+ and Ba2+. In contrast, trpl encodes a Ca2+-permeable cation channel that is constitutively active, not affected by thapsigargin, blocked by high micromolar Gd3+, and non-selective with respect to Ca2+, Na+, and Ba2+. The region of lowest sequence identity between Trp and Trpl occurs in the COOH-terminal domain. To test the hypothesis that this region is responsible for the differential sensitivity of these channels to thapsigargin, chimeric constructs of Trp and Trpl were created in which the COOH-terminal tail region of each protein was exchanged. The Trp construct with the Trpl COOH-tail was constitutively active, insensitive to thapsigargin, but retained selectivity for Ca2+ over Na+ and Ba2+. In contrast, the Trpl construct with the Trp COOH-tail was not constitutively active, could be activated by thapsigargin, but remained non-selective with respect to Ca2+, Ba2+, and Na+. These results suggest that the COOH-terminal domain of Trpl plays an important role in determining constitutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin.
Amino Acid Sequence↗
Receptor-coupled, DAG-gated Ca2+-permeable cationic channels in LNCaP human prostate cancer epithelial cells.
Although the prostate gland is a rich source of alpha1-adreno- (alpha1-AR) and m1-cholino receptors (m1-AChR), the membrane processes associated with their activation in glandular epithelial cells is poorly understood. We used the whole-cell patch-clamp technique to show that the agonists of the respective receptors, phenylephrine (PHE) and carbachol (CCh), activate cationic membrane currents in lymph node carcinoma of the prostate (LNCaP) human prostate cancer epithelial cells, which are not dependent on the filling status of intracellular IP3-sensitive Ca2+ stores, but directly gated by diacylglycerol (DAG), as evidenced by the ability of its membrane permeable analogue, OAG, to mimic the effects of the agonists. The underlying cationic channels are characterized by the weak field-strength Eisenman IV permeability sequence for monovalent cations (PK(25) > PCs(4.6) > PLi(1.4) > PNa(1.0)), and the following permeability sequence for divalent cations: PCa(1.0) > PMg(0.74) > PBa(0.6) > PSr(0.36) > PMn(0.3). They are 4.3 times more permeable to Ca2+ than Na+ and more sensitive to the inhibitor 2-APB than SK&F 96365. RT-PCR analysis shows that DAG-gated members of the transient receptor potential (TRP) channel family, including TRPC1 and TRPC3, are present in LNCaP cells. We conclude that, in prostate cancer epithelial cells, alpha1-ARs and m1-AChRs are functionally coupled to Ca2+-permeable DAG-gated cationic channels, for which TRPC1 and TRPC3 are the most likely candidates.
Base Sequence↗
Partial mitochondrial inhibition causes striatal dopamine release suppression and medium spiny neuron depolarization via H2O2 elevation, not ATP depletion.
Mitochondrial dysfunction is a potential causal factor in Parkinson's disease. We show here that acute exposure to the mitochondrial complex I inhibitor rotenone (30-100 nM; 30 min) causes concentration-dependent suppression of single-pulse evoked dopamine (DA) release monitored in real time with carbon-fiber microelectrodes in guinea pig striatal slices, with no effect on DA content. Suppression of DA release was prevented by the sulfonylurea glibenclamide, implicating ATP-sensitive K+ (KATP) channels; however, tissue ATP was unaltered. Because KATP channels can be activated by hydrogen peroxide (H2O2), as well as by low ATP, we examined the involvement of rotenone-enhanced H2O2 generation. Confirming an essential role for H2O2, the inhibition of DA release by rotenone was prevented by catalase, a peroxide-scavenging enzyme. Striatal H2O2 generation during rotenone exposure was examined in individual medium spiny neurons using fluorescence imaging with dichlorofluorescein (DCF). An increase in intracellular H2O2 levels followed a similar time course to that of DA release suppression and was accompanied by cell membrane depolarization, decreased input resistance, and increased excitability. Extracellular catalase markedly attenuated the increase in DCF fluorescence and prevented rotenone-induced effects on membrane properties; membrane changes were also largely prevented by flufenamic acid, a blocker of transient receptor potential (TRP) channels. Thus, partial mitochondrial inhibition can cause functional DA denervation via H2O2 and KATP channels, without DA or ATP depletion. Furthermore, amplified H2O2 levels and TRP channel activation in striatal spiny neurons indicate potential sources of damage in these cells. Overall, these novel factors could contribute to parkinsonian motor deficits and neuronal degeneration caused by mitochondrial dysfunction.
Adenosine Triphosphate↗
PGE2-induced apoptotic cell death in K562 human leukaemia cells.
Prostaglandin-E2 (PGE2) is known to trigger suicidal death of nucleated cells (apoptosis) and enucleated erythrocytes (eryptosis). In erythrocytes PGE2 induced suicidal cell death involves activation of nonselective cation channels leading to Ca2+ entry followed by cell shrinkage and triggering of Ca2+ sensitive cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. The present study was performed to explore whether PGE2 induces apoptosis of nucleated cells similarly through cation channel activation and to possibly disclose the molecular identity of the cation channels involved. To this end, Ca2+ activity was estimated from Fluo3 fluorescence, mitochondrial potential from DePsipher fluorescence, phosphatidylserine exposure from annexin binding, caspase activation from caspAce fluorescence, cell volume from FACS forward scatter, and DNA fragmentation utilizing a photometric enzyme immunoassay. Stimulation of K562 human leukaemia cells with PGE2 (50 microM) increased cytosolic Ca2+ activity, decreased forward scatter, depolarized the mitochondrial potential, increased annexin binding, led to caspase activation and resulted in DNA fragmentation. Gene silencing of the Ca2+-permeable transient receptor potential cation channel TRPC7 significantly blunted PGE2-induced triggering of PS exposure and DNA fragmentation. In conclusion, K562 cells express Ca2+-permeable TRPC7 channels, which are activated by PGE2 and participate in the triggering of apoptosis.
Acid Sensing Ion Channels↗
A C. elegans model of nicotine-dependent behavior: regulation by TRP-family channels.
Nicotine, the primary addictive substance in tobacco, induces profound behavioral responses in mammals, but the underlying genetic mechanisms are not well understood. Here we develop a C. elegans model of nicotine-dependent behavior. We show that worms exhibit behavioral responses to nicotine that parallel those observed in mammals, including acute response, tolerance, withdrawal, and sensitization. These nicotine responses require nicotinic acetylcholine receptor (nAChR) family genes that are known to mediate nicotine dependence in mammals, suggesting functional conservation of nAChRs in nicotine responses. Importantly, we find that mutant worms lacking TRPC (transient receptor potential canonical) channels are defective in their response to nicotine and that such a defect can be rescued by a human TRPC channel, revealing an unexpected role for TRPC channels in regulating nicotine-dependent behavior. Thus, C. elegans can be used to characterize known genes as well as to identify new genes regulating nicotine responses.
Amino Acid Sequence↗
NGF and GDNF differentially regulate TRPV1 expression that contributes to development of inflammatory thermal hyperalgesia.
The transient receptor potential ion channel, TRPV1 plays an essential role in the development of inflammatory thermal hyperalgesia. We investigated the dependence of inflammatory TRPV1 induction on neurotrophic factor. Rat dorsal root ganglia (DRG) neurons were classified according to immunostaining for trk-A and IB4 and the effects of antibodies against NGF or GDNF on TRPV1 expression within the groups were then analysed by immunohistochemical means. The data were compared with the time course of trophic factor expression and the effects of their antibodies on thermal hyperalgesia against radiant heat after inflammation. Although the levels of both NGF and GDNF were increased by inflammation, NGF rapidly and transiently increased whereas GDNF increased gradually over a period of approximately one week. TRPV1 expression was increased within both trk-A positive and IB4 positive neurons after inflammation. Increased TRPV1 expression within trk-A positive neurons was prevented by anti-NGF but not by anti-GDNF, whereas TRPV1 induction within the IB4 positive group was blocked by anti-GDNF but not by anti-NGF. Both antibodies prevented the short latency of withdrawing an inflamed paw from radiant heat. These results suggest that inflammation differentially increases both NGF and GDNF, which facilitate TRPV1 expression within distinctive neurons to induce thermal hyperalgesia.
Animals↗
Hyperforin activates nonselective cation channels (NSCCs).
A large body of evidence supports the preclinical antidepressant profile of hyperforin including inhibition of the synaptosomal uptake of several neurotransmitters by hyperforin and studies in behavioural models. In contrast to other antidepressants, hyperforin does not directly inhibit neurotransmitter transporters, but instead uptake inhibition seems to be the consequence of an elevated intracellular sodium concentration ([Na+]i). The mechanism of hyperforin-induced elevation of [Na+]i was investigated using two different cell types: human platelets and rat pheochromocytoma cells (PC12 cells). In both cell systems, hyperforin increased both [Na+]i and free intracellular Ca2+ concentration ([Ca2+]i). One pathway for Na+ and Ca2+ entry is mediated by nonselective cation channels (NSCCs), which can be blocked by SK&F 96365 and LOE 908. LOE 908 is a blocker of both NSCC1 and NSCC2 subclasses, while SK&F 96365 blocks NSCC2 only. Both SK&F 96365 and LOE 908 completely inhibited the hyperforin-induced influx of Na+ and Ca2+ into platelets and PC12 cells. This indicates that hyperforin is mainly active upon NSCC2. The effect of hyperforin is inhibited by La3+ and Gd3+, indicating that there is a potential homology with canonical transient receptor potential protein channels (TRPC channels). Moreover, La3+ and Gd3+ attenuate the effect of hyperforin on serotonin uptake in human platelets. Additionally, hyperforin induces barium influx in PC12 cells and this influx can be inhibited by SK&F 96365, LOE 908, Gd3+ and La3+. In summary, these findings suggest that hyperforin represents a new principle for preclinical antidepressant activity, modulating brain neurotransmission by inhibition of neurotransmitter uptake via activation of NSCCs.British Journal of Pharmacology (2005) 145, 75-83. doi:10.1038/sj.bjp.0706155.
Animals↗
Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion.
The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+O,-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trp1 cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2-17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and A1F4-, but not guanosine 5'-[beta-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] of FA1F4-. Staurosporine largely prevented the inhibition of store-activated Ca2+ inflow by GTP[S]. It is concluded that membrane fusion processes are unlikely to be involved in the link between the release of Ca2+ from the endoplasmic reticulum and activation of SACCs. The idea that this link is achieved by direct interaction of a protein(s) in the endoplasmic reticulum membrane with the SACC protein is briefly discussed.
Aniline Compounds↗
Involvement of a calcium-dependent dephosphorylation of BAD associated with the localization of Trpc-1 within lipid rafts in 7-ketocholesterol-induced THP-1 cell apoptosis.
7-Ketocholesterol is a component of oxidized LDL, which plays a central role in atherosclerosis. It is a potent inducer of cell death towards a wide number of cells involved in atherosclerosis. In this study, it is reported that 7-ketocholesterol treatment induces an increase of cytosolic-free Ca(2+) in THP-1 monocytic cells. This increase is correlated with the induction of cytotoxicity as suggested from experiments using the Ca(2+) channel blockers verapamil and nifedipine. This 7-ketocholesterol-induced apoptosis appears to be associated with the dephosphorylation of serine 75 and serine 99 of the proapoptotic protein Bcl-2 antagonist of cell death (BAD). We demonstrated that this dephosphorylation results mainly from the activation of calcium-dependent phosphatase calcineurin by the oxysterol-induced increase in Ca(2+). Moreover, this Ca(2+) increase appears related to the incorporation of 7-ketocholesterol into lipid raft domains of the plasma membrane, followed by the translocation of transient receptor potential calcium channel 1, a component of the store operated Ca(2+) entry channel, to rafts.
Apoptosis↗