Lysophosphatidylcholine (LPC) is a bioactive lipid present at high concentrations in inflamed and injured tissues where it contributes to the initiation and maintenance of pain. One of its important molecular effectors is the transient receptor potential canonical 5 (TRPC5), but the explicit mechanism of the activation is unknown. Using electrophysiology, mutagenesis and molecular dynamics simulations, we show that LPC-induced activation of TRPC5 is modulated by xanthine ligands and depolarizing voltage, and involves conserved residues within the lateral fenestration of the pore domain. Replacement of W577 with alanine (W577A) rendered the channel insensitive to strong depolarizing voltage, but LPC still activated this mutant at highly depolarizing potentials. Substitution of G606 located directly opposite position 577 with tryptophan rescued the sensitivity of W577A to depolarization. Molecular simulations showed that depolarization widens the lower gate of the channel and this conformational change is prevented by the W577A mutation or removal of resident lipids. We propose a gating scheme in which depolarizing voltage and lipid-pore helix interactions act together to promote TRPC5 channel opening.

• MeSH
• gating iontového kanálu účinky léků   MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPC * metabolismus   genetika   chemie   MeSH
• lidé MeSH
• lysofosfatidylcholiny * metabolismus   farmakologie   MeSH
• lysofosfolipidy metabolismus   farmakologie   MeSH
• membránové potenciály účinky léků   MeSH
• mutace MeSH
• simulace molekulární dynamiky * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The human transient receptor potential canonical 5 (TRPC5) is a calcium-permeable, nonselective cation channel expressed in the central and peripheral nervous system and also in other tissues such as the kidney, synovium, and odontoblasts. TRPC5 has been recently confirmed to play a key role in spontaneous, inflammatory mechanical, and cold pain. Although TRPC5 activation is known to be cold sensitive, it is unclear whether this property is intrinsic to the channel protein and whether or to what extent it may be determined by the cellular environment. In this study, we explored the cold sensitivity of human TRPC5 at the single-channel level using transiently transfected HEK293T cells. Upon decreasing the temperature, the channel demonstrated prolonged mean open dwell times and a robust increase in the open probability (Po ), whereas the amplitude of unitary currents decreased ~1.5-fold per 10°C of temperature difference. In the absence of any agonists, the temperature dependence of Po was sigmoidal, with a steep slope within the temperature range of 16°C-11°C, and exhibited saturation below 8-5°C. Thermodynamic analysis revealed significant changes in enthalpy and entropy, suggesting that substantial conformational changes accompany cold-induced gating. The mutant channel T970A, in which the regulation downstream of G-protein coupled receptor signaling was abrogated, exhibited higher basal activity at room temperature and a less steep temperature response profile, with an apparent threshold below 22°C. An even more pronounced decrease in the activation threshold was observed in a mutant that disrupted the electrostatic interaction of TRPC5 with the endoplasmic reticulum calcium sensor stromal interaction molecule 1. Thus, TRPC5 exhibits features of an intrinsically cold-gated channel; its sensitivity to cold tightly depends on the phosphorylation status of the protein and intracellular calcium homeostasis.

• MeSH
• buněčná membrána metabolismus   MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPC * genetika   metabolismus   MeSH
• lidé MeSH
• vápník * metabolismus   MeSH
• vápníkové kanály metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Transient receptor potential canonical 5 (TRPC5) is a polymodal, calcium-permeable, nonselective ion channel that is expressed in the brain and 75 % of human sensory neurons. Its pharmacological or genetic inhibition leads to the relief of neuropathic and inflammatory pain. The clinically approved drug duloxetine is superior to other serotonin and norepinephrine reuptake inhibitors at managing painful neuropathies, but it is not known why. Here we ask whether the TRPC5 receptor is modulated by duloxetine and may contribute to its analgesic effect. Electrophysiological measurements of heterologously expressed human TRPC5 in HEK293T cells were performed to evaluate the effect of duloxetine. The interaction site was identified by molecular docking and molecular dynamics simulations in combination with point mutagenesis. We found that duloxetine inhibits TRPC5 in a concentration-dependent manner with a high potency (IC50 = 0.54 ± 0.03 μM). Our data suggest that duloxetine binds into a voltage sensor-like domain. For the interaction, Glu418 exhibited particular importance due to putative hydrogen bond formation. Duloxetine effectively inhibits TRPC5 currents induced by cooling, voltage, direct agonists and by the stimulation of the PLC pathway. The finding that this TRPC5 inhibitor is widely used and well tolerated provides a scaffold for new pain treatment strategies.

• MeSH
• bolest * MeSH
• duloxetinum hydrochlorid farmakologie   MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPC * genetika   metabolismus   MeSH
• lidé MeSH
• simulace molekulového dockingu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

AIM: The pathogenesis of obesity has been associated with high intake of dietary fat, and some recent studies have explored the cellular mechanisms of oro-sensory detection of dietary fatty acids. We further assessed the role of transient receptor potential canonical (TRPC) channels in oro-sensory perception of dietary lipids. METHODS: We determined by RT-qPCR and western blotting the expression of TRPC3/6/7 channels in mouse fungiform taste bud cells (mTBC). Immunocytochemistry was used to explore whether TRPC3 channels were co-expressed with fatty acid receptors. We employed wild-type (WT) mTBC, and those transfected with small interfering RNAs (siRNAs) against TRPC3 or STIM1. Ca2+ signalling was studied in TBC from TRPC3-/- mice and their WT littermates. RESULTS: We demonstrate that mouse fungiform taste bud cells (mTBC) express TRPC3, but not TRPC6 or TRPC7 channels, and their inactivation by siRNA or experiments on TBC from TRPC3-/- mice brought about a decrease in fatty acid-induced gustatory Ca2+ signalling, coupled with taste bud CD36 lipid sensor. TRPC3 channel activation was found to be under the control of STIM1 in lingual mTBC. Behavioural studies showed that spontaneous preference for a dietary long-chain fatty acid was abolished in TRPC3-/- mice, and in mice wherein lingual TRPC3 expression was silenced by employing siRNA. CONCLUSION: We report that lingual TRPC3 channels are critically involved in fat taste perception.

• MeSH
• chuťová percepce * MeSH
• dietní tuky MeSH
• kationtové kanály TRPC genetika   MeSH
• lipidy MeSH
• myši MeSH
• preference v jídle * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

• MeSH
• anizotropie MeSH
• cirkulární dichroismus MeSH
• interakční proteinové domény a motivy MeSH
• kationtové kanály TRPC chemie   genetika   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• proteiny S100 chemie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• vápník chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

All secretory anterior pituitary cells fire action potentials spontaneously and exhibit a high resting cation conductance, but the channels involved in the background permeability have not been identified. In cultured lactotrophs and immortalized GH(3) cells, replacement of extracellular Na(+) with large organic cations, but not blockade of voltage-gated Na(+) influx, led to an instantaneous hyperpolarization of cell membranes that was associated with a cessation of spontaneous firing. When cells were clamped at -50 mV, which was close to the resting membrane potential in these cells, replacement of bath Na(+) with organic cations resulted in an outward-like current, reflecting an inhibition of the inward holding membrane current and indicating loss of a background-depolarizing conductance. Quantitative RT-PCR analysis revealed the high expression of mRNA transcripts for TRPC1 and much lower expression of TRPC6 in both lactotrophs and GH(3) cells. Very low expression of TRPC3, TRPC4, and TRPC5 mRNA transcripts were also present in pituitary but not GH(3) cells. 2-APB and SKF-96365, relatively selective blockers of TRPC channels, inhibited electrical activity, Ca(2+) influx and prolactin release in a concentration-dependent manner. Gd(3+), a common Ca(2+) channel blocker, and flufenamic acid, an inhibitor of non-selective cation channels, also inhibited electrical activity, Ca(2+) influx and prolactin release. These results indicate that nonselective cation channels, presumably belonging to the TRPC family, contribute to the background depolarizing conductance and firing of action potentials with consequent contribution to Ca(2+) influx and hormone release in lactotrophs and GH(3) cells.

• MeSH
• akční potenciály MeSH
• časové faktory MeSH
• iontové kanály genetika   metabolismus   účinky léků   MeSH
• kationtové kanály TRPC genetika   metabolismus   účinky léků   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• laktotropní buňky metabolismus   sekrece   účinky léků   MeSH
• messenger RNA metabolismus   MeSH
• metoda terčíkového zámku MeSH
• modulátory membránového transportu farmakologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• potkani Sprague-Dawley MeSH
• prolaktin sekrece   MeSH
• sodík metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

Blood filtration and formation of primary urine in the kidney glomerulus is provided by a specialized membrane called slit diaphragm located between well-branched pedicels of podocytes. Actually, the slit diaphragm is a protein supercomplex, whose disruption can cause failure of renal filtration, and patients usually manifest nephrotic syndrome. Recently, familial forms of nephrotic syndrome have been described which arise from malfunction of mutated proteins making up the slit diaphragm. In 2005 it was found that one of the proteins present in this complex was non-selective cation channel TRPC6. The aim of this work was to screen mutations and polymorphisms of the TRPC6 gene in a group of 64 Czech patients with nephrotic syndrome and subsequently, on the basis of these data, evaluate the role of mutations in the TRPC6 gene in Czech population. The analysis was performed by the PCR method followed by direct sequencing and high-resolution melting method. We have not identified any mutations in our group of patients. Two additional single nucleotide polymorphisms – p.P15S and p.A404V – were detected along with nucleotide changes that did not result in amino acid changes and with a few intronic changes. P.P15S heterozygotes were more frequent in patients with steroid-resistant FSGS than in steroid- sensitive patients (29 % versus 12.1 %). To conclude, we did not find any probable disease-causing mutation in the TRPC6 gene in the cohort of 64 Czech patients. The p.P15S polymorphism might have some influence on the therapeutic response of FSGS patients.

• MeSH
• dospělí MeSH
• fokálně segmentální glomeruloskleróza genetika   MeSH
• kationtové kanály TRPC genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipoidní nefróza genetika   MeSH
• mladý dospělý MeSH
• mutace MeSH
• mutační analýza DNA MeSH
• polymerázová řetězová reakce MeSH
• polymorfismus genetický MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

The transient receptor potential channel TRPC6 is a non-selective cation channel which modulates the calcium level in eukaryotic cells (including sensory receptor cells) in response to external signals. Calmodulin (CaM) is a ubiquitously expressed Ca(2+) binding protein that is an important mediator of Ca(2+)-dependent regulation of the TRPC6 channel. One CaM binding site was identified within the C-tail of TRPC6. The aim of this study is to map in detail the CaM and inositol (1,4,5)-triphosphate receptor binding (CIRB) domain in the C-terminal region of mouse TRPC6 that is capable of interacting with CaM using in vitro binding assays. Besides the set of positively charged amino acid residues Arg852, Lys856, Arg864, Lys859/Arg860, a hydrophobic Ile857, at the position 1 in 1-5-10 motif, was located and the effect of replacing it with a neutral residue was tested using fluorescence anisotropy measurement. Participation of Ile857 could indicate a strong role of this conserved CaM binding motif.

• MeSH
• fluorescenční polarizace MeSH
• kalmodulin metabolismus   MeSH
• kationtové kanály TRPC chemie   genetika   metabolismus   MeSH
• klonování DNA MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• myši MeSH
• retardační test MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hypoxic pulmonary vasoconstriction (HPV) is an important homeostatic mechanism in which increases of [Ca2+]i are primary events. In this study, primary cultured, human pulmonary artery smooth muscle cells (hPASMC) were used to examine the role of TRPC channels in mediating [Ca2+]i elevations during hypoxia. Hypoxia (PO2 about 20 mm Hg) evoked a transient [Ca2+]i elevation that was reduced by removal of extracellular calcium. Nifedipine and verapamil, blockers of voltage-gated calcium channels (VGCCs), attenuated the hypoxia-induced [Ca2+]i elevation by about 30 %, suggesting the presence of alternate Ca2+ entry pathways. Expression of TRPC1 and TRPC6 in hPASMC were found by RT-PCR and confirmed by Western blot analysis. Antagonists for TRPC, 2APB and SKF96365, significantly reduced hypoxia-induced [Ca2+]i elevation by almost 60 %. Both TRPC6 and TRPC1 were knocked down by siRNA, the loss of TRPC6 decreased hypoxic response down to 21 % of control, whereas the knockdown of TRPC1 reduced the hypoxia response to 85 %, suggesting that TRPC6 might play a central role in mediating hypoxia response in hPASMC. However, blockade of PLC pathway caused only small inhibition of the hypoxia response. In contrast, AICAR, the agonist of AMP-activated kinase (AMPK), induced a gradual [Ca2+]i elevation, whereas compound C, an antagonist of AMPK, almost abolished the hypoxia response. However, co-immunoprecipitation revealed that AMPK? was not colocalized with TRPC6. Our data supports a role for TRPC6 in mediation of the [Ca2+]i elevation in response to hypoxia in hPASMC and suggests that this response may be linked to cellular energy status via an activation of AMPK.

• MeSH
• arteria pulmonalis cytologie   fyziologie   MeSH
• financování organizované MeSH
• hypoxie buňky MeSH
• kationtové kanály TRPC genetika   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• myocyty hladké svaloviny cytologie   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteinkinasy aktivované AMP metabolismus   MeSH
• svaly hladké cévní cytologie   metabolismus   MeSH
• vápník metabolismus   MeSH
• vápníkové kanály metabolismus   MeSH
• Check Tag
• lidé MeSH