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PubMed2005

Functional role of TRPC channels in the regulation of endothelial permeability.

The endothelial cells (ECs) form a semipermeable barrier between the blood and the tissue. An important function of the endothelium is to maintain the integrity of the barrier function of the vessel wall. Ca(2+) signaling in ECs plays a key role in maintaining the barrier integrity. Transient receptor potential canonical (TRPC) channels are mammalian homologs of Drosophila TRP Ca(2+)-permeable channels expressed in EC. TRPC channels are thought to function as a Ca(2+) entry channel operated by store-depletion as well as receptor-activated channels in a variety of cell types, including ECs. Inflammatory mediators such as thrombin, histamine, bradykinin, and others increase endothelial permeability by actin polymerization-dependent EC rounding and formation of inter-endothelial gaps, a process critically dependent on the increase in EC cytosolic [Ca(2+)] ([Ca(2+)](i)). Increase in endothelial permeability depends on both intracellular Ca(2+) release and extracellular Ca(2+) entry through TRPC channels. This review summarizes recent findings on the role of TRPC channels in the mechanism of Ca(2+) entry in ECs, and, in particular, the role of TRPC channels in regulating endothelial barrier function.

Animals↗
PubMed2006

Focal and segmental glomerulosclerosis: varying biologic mechanisms underlie a final histopathologic end point.

Focal and segmental glomerulosclerosis (FSGS) is a pathologic entity that is a common and increasing cause of end-stage renal disease. Typical manifestations include proteinuria, hypertension, worsening renal insufficiency, and, frequently, renal failure. The etiology, however, remains unknown in a majority of patients. There is an estimated recurrence rate of 30% to 40% in renal transplant patients, suggesting that the pathogenesis is not solely a result of intrinsic kidney disease. Although some of its characteristics have been reported, the precise identification of a circulating factor associated with FSGS has not been made. Remarkable progress has been made in recent years regarding biologic mechanisms surrounding FSGS and proteinuria. Insight into the pathogenesis of FSGS has been gained through the study of hereditary forms of FSGS and nephrotic syndromes. Mutations in cytoskeletal proteins that affect podocyte structure have been the target until recently. Here we review the current understanding of this glomerular disease and areas for future concentration.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

The TRPC3/6/7 subfamily of cation channels.

The mammalian transient receptor potential (TRP) proteins consist of a superfamily of Ca2+-permeant non-selective cation channels with structural similarities to Drosophila TRP. The TRP superfamily can be divided into three major families, among them the "canonical TRP" family (TRPC). The seven protein products of the mammalian TRPC family of genes (designated TRPC1-7) share in common the activation through PLC-coupled receptors and have been proposed to encode components of native store-operated channels in different cell types. In addition, the three members of the TRPC3/6/7 subfamily of TRPC channels can be activated by diacylglycerol analogs, providing a possible mechanism of activation of these channels by PLC-coupled receptors. This review summarizes the current knowledge about the mechanism of activation of the TRPC3/6/7 subfamily, as well as the potential role of these proteins as components of native Ca2+-permeant channels.

Animals↗
PubMedDate not supplied

FRET-based analysis of TRPC subunit stoichiometry.

By analogy to other cation channel subunits with six transmembrane-spanning domains, the seven members of the "classical" or "canonical" transient receptor potential channels (TRPC) family are believed to assemble into homo- or heterotetrameric complexes. These complexes have been verified by classical methods such as coimmunoprecipitation, crosslinking analysis or functional assays applying dominant negative pore mutants. More recently, fluorescence resonance energy transfer (FRET)-a measure for the close proximity of fluorescent molecules-has become instrumental in monitoring protein assembly in living cells. Here we demonstrate further possibilities and verification procedures of the FRET technology to test the assembly of ion channel subunits. Temporally and spatially resolved FRET imaging demonstrates an early assembly of TRPC subunits in the endoplasmic reticulum and the Golgi apparatus. Confocal FRET imaging verifies FRET signals over the plasma membrane at high spatial resolution. Taking advantage of the quantitative analysis of digital video imaging, we demonstrate that FRET between TRPC subunits is only poorly concentration-dependent. Moreover, a correlation between the efficiency of energy transfer and the molar ratio of the FRET donor to the acceptor was exploited to verify the tetrameric stoichiometry of TRPC complexes. Finally, we introduce a competition-FRET assay to test the ability of wild-type TRPC subunits to recruit fluorescent TRPC subunits into separate channel complexes.

Calcium Channels↗
PubMed1999

Search for oncogenic regulators in an autocrine tumor model using differential display PCR: identification of novel candidate genes including the calcium channel mtrp6.

A hemopoietic multistep tumor model, in which IL-3 dependent PB-3c mast cells, following expression of v-H-ras progress in vivo to IL-3 producing autocrine tumors has previously been established. Central for this oncogenic progression is a recessive step, which is reversible by cell fusion and leads to stabilization of IL-3 mRNA with concomitant activation of the autocrine loop. Comparing the IL-3 dependent PB-3c and the IL-3 autocrine V2D1 tumor cells with differential display PCR revealed 12 differentially expressed genes of which eight were upregulated and four downregulated in the tumor. They included four proteases (mouse mast cell protease 2, granzyme B, pepsinogen F and serine protease 1) and two metabolic enzymes (adenine phosphoribosyltransferase and fructose1,6-bisphosphatase). For validation, expression of the identified genes was tested in independent PB-3c precursor clones and their tumor derivatives. Expression of an endogenous retroviral IAP element and three unknown transcripts were consistently upregulated in all tumor lines. In somatic cell hybrids, two of these unknown cDNAs showed a dominant and one a recessive expression pattern. One transcript, expressed in the precursor but downregulated in the tumor cells, was cloned and identified as the murine calcium channel mtrp6.

Amino Acid Sequence↗
PubMed2004

Canonical transient receptor potential TRPC7 can function as both a receptor- and store-operated channel in HEK-293 cells.

Previous studies on the activation mechanism of canonical transient receptor potential (TRPC) channels have often produced conflicting conclusions. All seven have been shown to be activated by phospholipase C (PLC)-coupled receptors, but TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC7 have also been proposed to function as store-operated channels.(1)1Although PLC activation inevitably leads to activation of store-operated channels, in this report when we refer to PLC-activated channels, we mean those channels that are specifically activated by PLC independently of store depletion. In the case of TRPC3, the expression environment and the expression level appear to determine the mode of regulation. Evidence of a close structural relative of TRPC3, TRPC7, has been presented that this channel is activated by receptor activation or by store depletion. On the basis of previous findings for TRPC3, we reasoned that subtle differences in structure or expression conditions might account for the apparent distinct gating mechanisms of TRPC7. To reexamine the mode of activation of TRPC7, we stably and transiently transfected human embryonic kidney (HEK)-293 cells with cDNA encoding for human TRPC7. We examined the ability of a PLC-activating agonist and an intracellular Ca(2+) store-depleting agent to activate these channels. Our findings demonstrate that when transiently expressed in HEK-293 cells, TRPC7 forms channels that are activated by PLC-stimulating agonists, but not by Ca(2+) store depletion. However, when stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca(2+) store depletion or PLC activation. To our knowledge, this is the first demonstration of a channel protein that can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories regarding TRPC7 regulation.

Barium↗
PubMed2003

Evidence of a role for TRPC channels in VEGF-mediated increased vascular permeability in vivo.

Vascular endothelial growth factor (VEGF) increases vascular permeability by stimulating endothelial Ca(2+) influx. Here we provide evidence that links VEGF-mediated increased permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) with diacylglycerol (DAG)-mediated activation of the transient receptor potential channels (TRPCs). We used the Landis-Michel technique to measure changes in hydraulic conductivity (L(p)) and fluorescence photometry to quantify changes in endothelial [Ca(2+)](i) in individually perfused Rana mesenteric microvessels in vivo and transfected nonendothelial cells in vitro. The membrane-permeant DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 microM), which is known to increase Ca(2+) influx through TRPCs, transiently increased L(p) 3.8 +/- 1.2-fold (from 1.6 +/- 0.8 to 9.8 +/- 2.7 x 10(-7) cm.s(-1).cmH(2)O(-1); P < 0.0001; n = 18). Protein kinase C inhibition by bisindolylmaleimide (1 microM) did not affect the OAG-induced increases in L(p). OAG also significantly increased microvascular endothelial [Ca(2+)](i) in vivo (n = 13; P < 0.0001), which again was not sensitive to protein kinase C inhibition. VEGF induced a transient increase in endothelial [Ca(2+)](i) in human embryonic kidney cells (HEK-293) that were cotransfected with VEGF receptor 2 and TRPC-6 but not with control, VEGF receptor 2, or TRPC-6 expression vector alone (P < 0.01; n = 9). Flufenamic acid, which has been shown to enhance activity of TRPC-6 but inhibit TRPC-3 and -7, enhanced the VEGF-mediated increase in L(p) in approximately half of the vessels tested but inhibited the response in the other half of the vessels. These data provide evidence consistent with the hypothesis that VEGF increases vascular permeability via DAG-mediated Ca(2+) entry through TRPCs. Although the exact identities of the TRPCs remain to be confirmed, TRPC-6 appears to be a likely candidate in approximately half of the vessels.

Animals↗
PubMed2003

Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle.

Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+]i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid (10 microM) and nifedipine (5 microM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+]i whereas MnCl2 (200 microM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 microM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.

Animals↗
PubMed2006

Expression of canonical transient receptor potential (TRPC) proteins in human glomerular mesangial cells.

Mesangial cells are located within glomerular capillary loops and contribute to the physiological regulation of glomerular hemodynamics. The function of mesangial cells is controlled by a variety of ion channels in the plasma membrane, including nonselective cation channels, receptor-operated Ca2+ channels, and recently identified store-operated Ca2+ channels. Although the significance of these channels has been widely acknowledged, their molecular identities are still unknown. Recently, the members of the canonical transient receptor potential (TRPC) protein family have been demonstrated to behave as cation channels. The present study was performed to identify the isoforms of endogenous TRPC proteins in human mesangial cells (HMCs) and their interactions. Western blotting showed that TRPC1, 3, 4, and 6 were expressed in cultured HMCs. Consistently, immunofluorescent confocal microscopy revealed specific stainings for TRPC1, 3, 4, and 6 with predominant intracellular localization. However, TRPC5 and 7 were not detectable at protein level by either Western blotting or immunofluorescent staining. The expression of TRPC1, 3, 4, and 6 was also observed in rat and human glomeruli using fluorescent immunohistochemistry. Furthermore, coimmunoprecipitation experiments and immunofluorescent double staining displayed that TRPC1 had physical interaction with TRPC4 and 6, while no interactions were detected among other isoforms of TRPCs. Ca2+ fluorescent ratiometry measurement showed that store-operated Ca2+ entry in HMCs was significantly reduced by knocking down TRPC1, but enhanced by overexpressing TRPC1. These results suggest that HMCs specifically express isoforms of TRPC1, 3, 4, and 6 proteins. These isoforms of TRPCs might selectively assemble to form functional complexes.

Blotting, Western↗
PubMed2005

TRPC6 - a new podocyte gene involved in focal segmental glomerulosclerosis.

Hereditary kidney diseases have long been an enigma with respect to the identity of the mutated genes and the mechanisms by which they develop. Recently, the podocyte has been identified as a primary target in both genetic and acquired glomerular disorders. Mutations discovered by Winn et al. and Reiser et al. in the gene encoding TRPC6, a non-selective cation channel of the TRP family expressed in podocyte foot processes, have been shown to cause focal segmental glomerulosclerosis. It remains to be determined whether these mutations lead to (i) impaired channel function that initiates a new pathogenic mechanism or (ii) decreased ability of the podocyte to adapt to normal physiological challenges that account for disease development, as suggested for other late-onset autosomal-dominant podocyte disorders.

Glomerulosclerosis, Focal Segmental↗
PubMed2007

TRPC6.

TRPC6 is a Ca(2+)-permeable non-selective cation channel expressed in brain, smooth muscle containing tissues and kidney, as well as in immune and blood cells. Channel homomers heterologously expressed have a characteristic doubly rectifying current-voltage relationship and are six times more permeable for Ca2+ than for Na+. In smooth muscle tissues, however, Na+ influx and activation of voltage-gated calcium channels by membrane depolarization rather than Ca2+ elevation by TRPC6 channels is the driving force for contraction. TRPC6 channels are directly activated by the second messenger diacylglycerol (DAG) and regulated by specific tyrosine or serine phosphorylation. Extracellular Ca2+ has inhibitory effects, while Ca2+/calmodulin acting from the intracellular side has potentiator effects on channel activity. Given its specific expression, TRPC6 is likely to play a number of physiological roles. Studies with TRPC6(-/-) mice suggest a role for the channel in the regulation of vascular and pulmonary smooth muscle contraction. TRPC6 was identified as an essential component of the slit diaphragm architecture of kidney podocytes. Other functions in immune and blood cells, as well as in brain and in smooth muscle-containing tissues such as stomach, colon and myometrium, remain elusive.

Animals↗
PubMed2006

VEGF activates receptor-operated cation channels in human microvascular endothelial cells.

OBJECTIVE: Vascular endothelial growth factor (VEGF) exerts many of its effects by stimulating endothelial calcium influx, but little is known about channels mediating VEGF-induced cation entry. The aim of this study was to measure and characterize for the first time the VEGF-activated cation current in human microvascular endothelial cells (HMVECs). METHODS AND RESULTS: Whole-cell patch-clamp recordings were made from HMVECs. During applied voltage ramps, VEGF activated a current that reversed at 0 mV, was sensitive to gadolinium, and required extracellular cations. Noise analysis yielded a single-channel conductance of 27 pS. The current was not dependent on intracellular calcium stores, and was not blocked by inositol triphosphate (IP3) receptor or serine/threonine kinase inhibition but was partially inhibited by flufenamic acid. A similar current was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG). To determine whether VEGF could activate recombinant ion channels with similar properties, we investigated the effect of VEGF on Chinese hamster ovary cells cotransfected with VEGFR2 and the canonical transient receptor potential (TRPC) channels, TRPC3 or TRPC6. VEGF induced a similar current to that described above in VEGFR2-TRPC3 and VEGFR2-TRPC6 cells but not in cells transfected with either cDNA alone. CONCLUSIONS: VEGF activates a receptor-operated cation current in HMVECs and OAG can activate directly a similar current in these cells. VEGF is also able to activate heterologously expressed TRPC3/6 channels through VEGFR2.

Animals↗
PubMedDate not supplied

Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces the change of calcium mobilization via TRPC ion channels in cultured human corporal smooth muscle cells.

Hypercholesterolemia is a major risk factor for erectile dysfunction. To understand the mechanism(s) of hypercholesterolemia-induced erectile dysfunction, we studied the effect of lysophosphatidylcholine (LPC) on the membrane conductance of corporal smooth muscle cells. We used cultured human corporal smooth muscle cells. The intracelluar Ca2+ concentration ([Ca2+]i) and the influx of divalent cation was monitored by the ratio of fura-2 fluorescence (F(340/380)) and by the Mn2+-induced quenching rate of fura-2, respectively. The LPC-induced membrane current was characterized by the whole-cell patch-clamp technique and the molecular identity of suspected channels was probed by RT-PCR. LPC (20 microM) induced a statistically significant increase in F(340/380) to 119.9+/-3.9% of initial control (n=6) in corporal smooth muscle cells. The addition of 20 microM LPC accelerated the quenching rate of F360 by 59.5+/-11.8% (n=5). LPC activated nonselective cationic current (ILPC), similar to the known effects of phenylephrine in corporal myocytes. The size of ILPC at -60 mV was -55.3+/-6.3 pA (n=8). The transcript of transient receptor potential channel 6 (TRPC6) was detected in human corporal myocytes. We also found one splicing variant of TRPC6, TRPC6alpha. In conclusion, the present study suggests that the LPC, a major component of oxidized low-density lipoprotiens, increases calcium in corporal smooth muscle cells probably through activation of a TRPC6 channel and the increased [Ca2+]i by LPC via TRP channels is one of mechanisms for hypercholesterolemia-induced erectile dysfunction.

Alternative Splicing↗
PubMed2005

Transient receptor potential protein subunit assembly and membrane distribution in human platelets.

We have previously suggested that the human homologue of the Drosophila transient receptor potential protein, TRPC1, is involved in conducting store-operated Ca2+ entry (SOCE) in human platelets since an antibody raised against the pore-forming region of TRPC1 inhibited SOCE. Here we have investigated plasma membrane expression of TRPC1 in human platelets and have probed for the presence of other TRPC proteins in these cells. Biotinylation revealed the presence of TRPC1 in the plasma membrane of resting platelets. Surface expression was not detectibly changed following Ca2+ store depletion or stimulation with thrombin. Western blotting demonstrated the presence of TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 in platelet lysates. TRPC1, TRPC4 and TRPC5 coimmunoprecipitated, as did TRPC3 and TRPC6. TRPC1, TRPC4 and TRPC5 were associated with detergent-resistant platelet membranes, from which they were partially released when the cells were cholesterol-depleted using methyl-beta-cyclodextrin. The distributions of TRPC3 and TRPC6 between soluble and membrane fractions were not affected by methyl-beta-cyclodextrin treatment. These results suggest that TRPC1, TRPC4 and TRPC5 form a heteromultimer associated with platelet lipid raft domains, whereas TRPC3 and TRPC6 associate independently of lipid rafts.

Biotinylation↗
PubMed2004

Expression of TRPC homologs in endothelial cells and smooth muscle layers of human arteries.

TRPC channels are a group of Ca(2+)-permeable nonselective cation channels that mediate store-operated and/or agonist-stimulated Ca(2+) influx in a variety of cell types. In this study, we extensively examined the expression patterns of TRPC homologs in human vascular tissues. RT-PCR amplified cDNA fragments of TRPC1 (505 bp), TRPC3 (372 bp), TRPC4 (499 bp), TRPC5 (325 bp), TRPC6 (509 bp), and TRPC7 (187 bp) from RNA isolated from cultured human coronary artery endothelial cells. In situ hybridization yielded strong labeling of TRPC1,3-6 in the endothelial and smooth muscle cells of human coronary and cerebral arteries. TRPC7 labeling was exclusively found in endothelial cells but not in smooth muscle cells. Results from immunohistochemical staining were consistent with those from in situ hybridization. Similar expression patterns of TRPC homologs were also observed in arterioles and vaso vasora. In conclusion, our study indicates that TRPC homologs are widely expressed in human vessels of all calibers, including medium-sized coronary arteries and cerebral arteries, smaller-sized resistance arteries, and vaso vasora. These results suggest a ubiquitous role of TRPC homologs in regulating blood supply to different regions and in controlling arterial blood pressure.

Adolescent↗
PubMed2006

Human TRPC6 expressed in HEK 293 cells forms non-selective cation channels with limited Ca2+ permeability.

TRPC6 is thought to be a Ca(2+)-permeable cation channel activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C (PLC). TRPC6 current is also activated by exogenous application of 1-oleoyl-acetyl-sn-glycerol (OAG) or by inhibiting 1,2-diacylglycerol (DAG) lipase activity using RHC80267. In the present study, both OAG and RHC80267 increased whole-cell TRPC6 current in cells from a human embryonic kidney cell line (HEK 293) stably expressing TRPC6, but neither compound increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) when the cells were bathed in high-K(+) buffer to hold the membrane potential near 0 mV. These results suggested that TRPC6 channels have limited Ca(2+) permeability relative to monovalent cation permeability and/or that Ca(2+) influx via TRPC6 is greatly attenuated by depolarization. To evaluate Ca(2+) permeability, TRPC6 currents were examined in extracellular buffer in which Ca(2+) was varied from 0.02 to 20 mm. The results were consistent with a pore-permeation model in which Ca(2+) acts primarily as a blocking ion and contributes only a small percentage ( approximately 4%) to whole-cell currents in the presence of extracellular Na(+). Measurement of single-cell fura-2 fluorescence during perforated-patch recording of TRPC6 currents showed that OAG increased [Ca(2+)](i) 50-100 nm when the membrane potential was clamped at between -50 and -80 mV, but had little or no effect if the membrane potential was left uncontrolled. These results suggest that in cells exhibiting a high input resistance, the primary effect of activating TRPC6 will be membrane depolarization. However, in cells able to maintain a hyperpolarized potential (e.g. cells with a large inwardly rectifying or Ca(2+)-activated K(+) current), activation of TRPC6 will lead to a sustained increase in [Ca(2+)](i). Thus, the contribution of TRPC6 current to both the kinetics and magnitude of the Ca(2+) response will be cell specific and dependent upon the complement of other channel types.

Calcium↗