RESUMO
Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.
Assuntos
Acetilcolina/imunologia , Proteínas de Bactérias/farmacologia , Cílios/imunologia , Depuração Mucociliar/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Canais de Cátion TRPM/imunologia , Traqueia/imunologia , Acetilcolina/metabolismo , Animais , Proteínas de Bactérias/imunologia , Transporte Biológico , Cílios/efeitos dos fármacos , Cílios/metabolismo , Feminino , Formiatos/metabolismo , Expressão Gênica , Humanos , Imunidade Inata , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Optogenética/métodos , Comunicação Parácrina/imunologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Papilas Gustativas/imunologia , Papilas Gustativas/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/patologia , VirulênciaRESUMO
Mutations in the PKD2 gene, which encodes the polycystin-2 (PC2, also called TRPP2) protein, lead to autosomal dominant polycystic kidney disease (ADPKD). As a member of the transient receptor potential (TRP) channel superfamily, PC2 functions as a non-selective cation channel. The activation and regulation of the PC2 channel are largely unknown, and direct binding of small-molecule ligands to this channel has not been reported. In this work, we found that most known small-molecule agonists of the mucolipin TRP (TRPML) channels inhibit the activity of the PC2_F604P, a gain-of-function mutant of the PC2 channel. However, two of them, ML-SA1 and SF-51, have dual regulatory effects, with low concentration further activating PC2_F604P, and high concentration leading to inactivation of the channel. With two cryo-electron microscopy (cryo-EM) structures, a molecular docking model, and mutagenesis results, we identified two distinct binding sites of ML-SA1 in PC2_F604P that are responsible for activation and inactivation, respectively. These results provide structural and functional insights into how ligands regulate PC2 channel function through unusual mechanisms and may help design compounds that are more efficient and specific in regulating the PC2 channel and potentially also for ADPKD treatment.
Assuntos
Rim Policístico Autossômico Dominante , Canais de Cátion TRPP , Humanos , Canais de Cátion TRPP/metabolismo , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Microscopia Crioeletrônica , Simulação de Acoplamento Molecular , Canais IônicosRESUMO
Some tropical sea cucumbers of the family Holothuriidae can efficiently repel or even fatally ensnare predators by sacrificially ejecting a bioadhesive matrix termed the Cuvierian organ (CO), so named by the French zoologist Georges Cuvier who first described it in 1831. Still, the precise mechanisms for how adhesiveness genetically arose in CO and how sea cucumbers perceive and transduce danger signals for CO expulsion during defense have remained unclear. Here, we report the first high-quality, chromosome-level genome assembly of Holothuria leucospilota, an ecologically significant sea cucumber with prototypical CO. The H. leucospilota genome reveals characteristic long-repeat signatures in CO-specific outer-layer proteins, analogous to fibrous proteins of disparate species origins, including spider spidroin and silkworm fibroin. Intriguingly, several CO-specific proteins occur with amyloid-like patterns featuring extensive intramolecular cross-ß structures readily stainable by amyloid indicator dyes. Distinct proteins within the CO connective tissue and outer surface cooperate to give the expelled matrix its apparent tenacity and adhesiveness, respectively. Genomic evidence offers further hints that H. leucospilota directly transduces predator-induced mechanical pressure onto the CO surface through mediation by transient receptor potential channels, which culminates in acetylcholine-triggered CO expulsion in part or in entirety. Evolutionarily, innovative events in two distinct regions of the H. leucospilota genome have apparently spurred CO's differentiation from the respiratory tree to a lethal defensive organ against predators.
Assuntos
Holothuria , Pepinos-do-Mar , Animais , Holothuria/genética , Holothuria/química , Holothuria/metabolismo , Proteínas Amiloidogênicas/metabolismo , AdesividadeRESUMO
Upon its mucosal transmission, HIV type 1 (HIV-1) rapidly targets genital antigen-presenting Langerhans cells (LCs), which subsequently transfer infectious virus to CD4+ T cells. We previously described an inhibitory neuroimmune cross talk, whereby calcitonin gene-related peptide (CGRP), a neuropeptide secreted by peripheral pain-sensing nociceptor neurons innervating all mucosal epithelia and associating with LCs, strongly inhibits HIV-1 transfer. As nociceptors secret CGRP following the activation of their Ca2+ ion channel transient receptor potential vanilloid 1 (TRPV1), and as we reported that LCs secret low levels of CGRP, we investigated whether LCs express functional TRPV1. We found that human LCs expressed mRNA and protein of TRPV1, which was functional and induced Ca2+ influx following activation with TRPV1 agonists, including capsaicin (CP). The treatment of LCs with TRPV1 agonists also increased CGRP secretion, reaching its anti-HIV-1 inhibitory concentrations. Accordingly, CP pretreatment significantly inhibited LCs-mediated HIV-1 transfer to CD4+ T cells, which was abrogated by both TRPV1 and CGRP receptor antagonists. Like CGRP, CP-induced inhibition of HIV-1 transfer was mediated via increased CCL3 secretion and HIV-1 degradation. CP also inhibited direct CD4+ T cells HIV-1 infection, but in CGRP-independent manners. Finally, pretreatment of inner foreskin tissue explants with CP markedly increased CGRP and CCL3 secretion, and upon subsequent polarized exposure to HIV-1, inhibited an increase in LC-T cell conjugate formation and consequently T cell infection. Our results reveal that TRPV1 activation in human LCs and CD4+ T cells inhibits mucosal HIV-1 infection, via CGRP-dependent/independent mechanisms. Formulations containing TRPV1 agonists, already approved for pain relief, could hence be useful against HIV-1.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Infecções por HIV , Humanos , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Linfócitos T/metabolismo , Células de Langerhans/metabolismo , Mucosa/metabolismo , Capsaicina/farmacologia , Dor/metabolismo , Infecções por HIV/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
Cellular zinc ions (Zn2+) are crucial for signal transduction in various cell types. The transient receptor potential (TRP) ankyrin 1 (TRPA1) channel, known for its sensitivity to intracellular Zn2+ ([Zn2+]i), has been a subject of limited understanding regarding its molecular mechanism. Here, we used metal ion-affinity prediction, three-dimensional structural modeling, and mutagenesis, utilizing data from the Protein Data Bank and AlphaFold database, to elucidate the [Zn2+]i binding domain (IZD) structure composed by specific AAs residues in human (hTRPA1) and chicken TRPA1 (gTRPA1). External Zn2+ induced activation in hTRPA1, while not in gTRPA1. Moreover, external Zn2+ elevated [Zn2+]i specifically in hTRPA1. Notably, both hTRPA1 and gTRPA1 exhibited inherent sensitivity to [Zn2+]i, as evidenced by their activation upon internal Zn2+ application. The critical AAs within IZDs, specifically histidine at 983/984, lysine at 711/717, tyrosine at 714/720, and glutamate at 987/988 in IZD1, and H983/H984, tryptophan at 710/716, E854/E855, and glutamine at 979/980 in IZD2, were identified in hTRPA1/gTRPA1. Furthermore, mutations, such as the substitution of arginine at 919 (R919) to H919, abrogated the response to external Zn2+ in hTRPA1. Among single-nucleotide polymorphisms (SNPs) at Y714 and a triple SNP at R919 in hTRPA1, we revealed that the Zn2+ responses were attenuated in mutants carrying the Y714 and R919 substitution to asparagine and proline, respectively. Overall, this study unveils the intrinsic sensitivity of hTRPA1 and gTRPA1 to [Zn2+]i mediated through IZDs. Furthermore, our findings suggest that specific SNP mutations can alter the responsiveness of hTRPA1 to extracellular and intracellular Zn2+.
Assuntos
Galinhas , Canal de Cátion TRPA1 , Zinco , Zinco/metabolismo , Zinco/química , Humanos , Canal de Cátion TRPA1/metabolismo , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/química , Animais , Células HEK293 , Domínios Proteicos , Especificidade da EspécieRESUMO
Polycystin-2 (PC2) is mutated in â¼15% of patients with autosomal dominant polycystic kidney disease (ADPKD). PC2 belongs to the family of transient receptor potential (TRP) channels and can function as a homotetramer. We investigated whether three disease-associated mutations (F629S, C632R, or R638C) localized in the channel's pore loop alter ion channel properties of human PC2 expressed in Xenopus laevis oocytes. Expression of wild-type (WT) PC2 typically resulted in small but measurable Na+ inward currents in the absence of extracellular divalent cations. These currents were no longer observed when individual pore mutations were introduced in WT PC2. Similarly, Na+ inward currents mediated by the F604P gain-of-function (GOF) PC2 construct (PC2 F604P) were abolished by each of the three pore mutations. In contrast, when the mutations were introduced in another GOF construct, PC2 L677A N681A, only C632R had a complete loss-of-function effect, whereas significant residual Na+ inward currents were observed with F629S (â¼15%) and R638C (â¼30%). Importantly, the R638C mutation also abolished the Ca2+ permeability of PC2 L677A N681A and altered its monovalent cation selectivity. To elucidate the molecular mechanisms by which the R638C mutation affects channel function, molecular dynamics (MD) simulations were used in combination with functional experiments and site-directed mutagenesis. Our findings suggest that R638C stabilizes ionic interactions between Na+ ions and the selectivity filter residue D643. This probably explains the reduced monovalent cation conductance of the mutant channel. In summary, our data support the concept that altered ion channel properties of PC2 contribute to the pathogenesis of ADPKD.
Assuntos
Mutação de Sentido Incorreto , Canais de Cátion TRPP , Xenopus laevis , Animais , Humanos , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Canais de Cátion TRPP/química , Sódio/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Oócitos/metabolismoRESUMO
Diabetic retinopathy (DR) is a common microvascular complication that causes visual impairment or loss. Aquaporin 4 (AQP4) is a regulatory protein involved in water transport and metabolism. In previous studies, we found that AQP4 is related to hypoxia injury in Muller cells. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a non-selective cation channel protein involved in the regulation of a variety of ophthalmic diseases. However, the effects of AQP4 and TRPV4 on ferroptosis and oxidative stress in high glucose (HG)-treated Muller cells are unclear. In this study, we investigated the functions of AQP4 and TRPV4 in DR. HG was used to treat mouse Muller cells. Reverse transcription quantitative polymerase chain reaction was used to measure AQP4 mRNA expression. Western blotting was used to detect the protein levels of AQP4, PTGS2, GPX4, and TRPV4. Cell count kit-8, flow cytometry, 5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolyl carbocyanine iodide staining, and glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits were used to evaluate the function of the Muller cells. Streptozotocin was used to induce DR in rats. Haematoxylin and eosin staining was performed to stain the retina of rats. GSH, SOD, and MDA detection kits, immunofluorescence, and flow cytometry assays were performed to study the function of AQP4 and TRPV4 in DR rats. Results found that AQP4 and TRPV4 were overexpressed in HG-induced Muller cells and streptozotocin-induced DR rats. AQP4 inhibition promoted proliferation and cell cycle progression, repressed cell apoptosis, ferroptosis, and oxidative stress, and alleviated retinal injury in DR rats. Mechanistically, AQP4 positively regulated TRPV4 expression. Overexpression of TRPV4 enhanced ferroptosis and oxidative stress in HG-treated Muller cells, and inhibition of TRPV4 had a protective effect on DR-induced retinal injury in rats. In conclusion, inhibition of AQP4 inhibits the ferroptosis and oxidative stress in Muller cells by downregulating TRPV4, which may be a potential target for DR therapy.
Assuntos
Aquaporina 4 , Retinopatia Diabética , Células Ependimogliais , Ferroptose , Estresse Oxidativo , Canais de Cátion TRPV , Animais , Masculino , Camundongos , Ratos , Aquaporina 4/metabolismo , Aquaporina 4/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Retinopatia Diabética/genética , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Glucose/metabolismo , Glucose/farmacologia , Camundongos Endogâmicos C57BL , Ratos Sprague-Dawley , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genéticaRESUMO
Idiopathic pulmonary fibrosis (IPF) poses significant challenges due to limited treatment options despite its complex pathogenesis involving cellular and molecular mechanisms. This study investigated the role of transient receptor potential ankyrin 1 (TRPA1) channels in regulating M2 macrophage polarization in IPF progression, potentially offering novel therapeutic targets. Using a bleomycin-induced pulmonary fibrosis model in C57BL/6J mice, we assessed the therapeutic potential of the TRPA1 inhibitor HC-030031. TRPA1 upregulation was observed in fibrotic lungs, correlating with worsened lung function and reduced survival. TRPA1 inhibition mitigated fibrosis severity, evidenced by decreased collagen deposition and restored lung tissue stiffness. Furthermore, TRPA1 blockade reversed aberrant M2 macrophage polarization induced by bleomycin, associated with reduced Smad2 phosphorylation in the TGF-ß1-Smad2 pathway. In vitro studies with THP-1 cells treated with bleomycin and HC-030031 corroborated these findings, highlighting TRPA1's involvement in fibrotic modulation and macrophage polarization control. Overall, targeting TRPA1 channels presents promising therapeutic potential in managing pulmonary fibrosis by reducing pro-fibrotic marker expression, inhibiting M2 macrophage polarization, and diminishing collagen deposition. This study sheds light on a novel avenue for therapeutic intervention in IPF, addressing a critical need in the management of this challenging disease.
Assuntos
Fibrose Pulmonar Idiopática , Macrófagos , Canal de Cátion TRPA1 , Animais , Camundongos , Acetanilidas , Bleomicina , Colágeno , Proteínas do Citoesqueleto , Camundongos Endogâmicos C57BL , Purinas , Canal de Cátion TRPA1/metabolismoRESUMO
The Ca2+ and ADP ribose (ADPR)-activated cation channel TRPM2 is the closest homolog of the cold sensor TRPM8 but serves as a deep-brain warmth sensor. To unravel the molecular mechanism of heat sensing by the TRPM2 protein, we study here temperature dependence of TRPM2 currents in cell-free membrane patches across ranges of agonist concentrations. We find that channel gating remains strictly agonist-dependent even at 40°C: heating alone or in combination with just Ca2+, just ADPR, Ca2+ + cyclic ADPR, or H2O2 pretreatment only marginally activates TRPM2. For fully liganded TRPM2, pore opening is intrinsically endothermic, due to ~10-fold larger activation enthalpy for opening (~200 kJ/mol) than for closure (~20 kJ/mol). However, the temperature threshold is too high (>40°C) for unliganded but too low (<15°C) for fully liganded channels. Thus, warmth sensitivity around 37°C is restricted to narrow ranges of agonist concentrations. For ADPR, that range matches, but for Ca2+, it exceeds bulk cytosolic values. The supraphysiological [Ca2+] needed for TRPM2 warmth sensitivity is provided by Ca2+ entering through the channel's pore. That positive feedback provides further strong amplification to the TRPM2 temperature response (Q10 ~ 1,000), enabling the TRPM2 protein to autonomously respond to tiny temperature fluctuations around 37°C. These functional data together with published structures suggest a molecular mechanism for opposite temperature dependences of two closely related channel proteins.
Assuntos
Canais de Cátion TRPM , Canais de Cátion TRPM/metabolismo , Temperatura Alta , Peróxido de Hidrogênio/metabolismo , Cálcio/metabolismo , Adenosina Difosfato Ribose/metabolismoRESUMO
Acute nociception is essential for survival by warning organisms against potential dangers, whereas tissue injury results in a nociceptive hypersensitivity state that is closely associated with debilitating disease conditions, such as chronic pain. Transient receptor potential (Trp) ion channels expressed in nociceptors detect noxious thermal and chemical stimuli to initiate acute nociception. The existing hypersensitivity model suggests that under tissue injury and inflammation, the same Trp channels in nociceptors are sensitized through transcriptional and posttranslational modulation, leading to nociceptive hypersensitivity. Unexpectedly and different from this model, we find that in Drosophila larvae, acute heat nociception and tissue injury-induced hypersensitivity involve distinct cellular and molecular mechanisms. Specifically, TrpA1-D in peripheral sensory neurons mediates acute heat nociception, whereas TrpA1-C in a cluster of larval brain neurons transduces the heat stimulus under the allodynia state. As a result, interfering with synaptic transmission of these brain neurons or genetic targeting of TrpA1-C blocks heat allodynia but not acute heat nociception. TrpA1-C and TrpA1-D are two splicing variants of TrpA1 channels and are coexpressed in these brain neurons. We further show that Gq-phospholipase C signaling, downstream of the proalgesic neuropeptide Tachykinin, differentially modulates these two TrpA1 isoforms in the brain neurons by selectively sensitizing heat responses of TrpA1-C but not TrpA1-D. Together, our studies provide evidence that nociception and noncaptive sensitization could be mediated by distinct sensory neurons and molecular sensors.
Assuntos
Nociceptividade , Canais de Potencial de Receptor Transitório , Animais , Drosophila/fisiologia , Neurônios , Nociceptividade/fisiologia , Nociceptores/fisiologia , Transdutores , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Transient receptor potential (TRP) melastatin member 8 (TRPM8), which is a calcium-permeable ion channel, functions as the primary molecular sensor of cold and menthol in humans. Recent cryoelectron microscopy (cryo-EM) studies of TRPM8 have shown distinct structural features in its architecture and domain assembly compared with the capsaicin receptor TRP vanilloid member 1 (TRPV1). Moreover, ligand-bound TRPM8 structures have uncovered unforeseen binding sites for both cooling agonists and membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. These complex structures unveil the molecular basis of cooling agonist sensing by TRPM8 and the allosteric role of PI(4,5)P2 in agonist binding for TRPM8 activation. Here, we review the recent advances in TRPM8 structural biology and investigate the molecular principles governing the distinguishing role of TRPM8 as the evolutionarily conserved menthol receptor.
Assuntos
Mentol , Canais de Cátion TRPM , Microscopia Crioeletrônica , Humanos , Ligantes , FosfatidilinositóisRESUMO
Small organic molecules in the intestinal lumen, particularly short-chain fatty acids (SCFAs) and glucose, have long been postulated to enhance calcium absorption. Here, we used 45Ca radioactive tracer to determine calcium fluxes across the rat intestine after exposure to glucose and SCFAs. Confirming previous reports, glucose was found to increase the apical-to-basolateral calcium flux in the cecum. Under apical glucose-free conditions, SCFAs (e.g., butyrate) stimulated the cecal calcium fluxes by approximately twofold, while having no effect on proximal colon. Since SCFAs could be absorbed into the circulation, we further determined whether basolateral SCFA exposure rendered some positive actions. It was found that exposure of duodenum and cecum on the basolateral side to acetate or butyrate increased calcium fluxes. Under butyrate-rich conditions, cecal calcium transport was partially diminished by Na+/H+ exchanger 3 (NHE3) inhibitor (tenapanor) and nonselective transient receptor potential vanilloid subfamily 6 (TRPV6) inhibitor (miconazole). To confirm the contribution of TRPV6 to SCFA-stimulated calcium transport, we synthesized another TRPV6 inhibitor that was demonstrated by in silico molecular docking and molecular dynamics to occlude TRPV6 pore and diminish the glucose- and butyrate-induced calcium fluxes. Therefore, besides corroborating the importance of luminal molecules in calcium absorption, our findings provided foundation for development of more effective calcium-rich nutraceuticals in combination with various absorptive enhancers, e.g., glucose and SCFAs.NEW & NOTEWORTHY Organic molecules in the intestinal lumen, e.g., glucose and short-chain fatty acids (SCFAs), the latter of which are normally produced by microfloral fermentation, can stimulate calcium absorption dependent on transient receptor potential vanilloid subfamily 6 (TRPV6) and Na+/H+ exchanger 3 (NHE3). A selective TRPV6 inhibitor synthesized and demonstrated by in silico docking and molecular dynamics to specifically bind to the pore domain of TRPV6 was used to confirm a significant contribution of this channel. Our findings corroborate physiological significance of nutrients and SCFAs in enhancing calcium absorption.
Assuntos
Cálcio , Ácidos Graxos Voláteis , Ratos , Animais , Trocador 3 de Sódio-Hidrogênio/metabolismo , Cálcio/metabolismo , Simulação de Acoplamento Molecular , Ácidos Graxos Voláteis/farmacologia , Ácidos Graxos Voláteis/metabolismo , Butiratos/farmacologia , Proteínas de Transporte/metabolismo , Duodeno/metabolismo , Glucose/metabolismo , Absorção IntestinalRESUMO
Cardiac arrhythmias pose a major threat to a patient's health, yet prove to be often difficult to predict, prevent and treat. A key mechanism in the occurrence of arrhythmias is disturbed Ca2+ homeostasis in cardiac muscle cells. As a Ca2+-activated non-selective cation channel, TRPM4 has been linked to Ca2+-induced arrhythmias, potentially contributing to translating an increase in intracellular Ca2+ concentration into membrane depolarisation and an increase in cellular excitability. Indeed, evidence from genetically modified mice, analysis of mutations in human patients and the identification of a TRPM4 blocking compound that can be applied in vivo further underscore this hypothesis. Here, we provide an overview of these data in the context of our current understanding of Ca2+-dependent arrhythmias.
RESUMO
We determined the role played by the transient receptor potential canonical 6 (TRPC6) channel in evoking the mechanical component of the exercise pressor reflex in male decerebrated Sprague-Dawley rats. TRPC6 channels were identified by quadruple-labelled (DiI, TRPC6, neurofilament-200 and peripherin) immunohistochemistry in dorsal root ganglion (DRG) cells innervating the triceps surae muscles (n = 12). The exercise pressor reflex was evoked by statically contracting the triceps surae muscles before and after injection of the TRPC6 antagonist BI-749327 (n = 11; 12 µg kg-1 ) or SAR7334 (n = 11; 7 µg kg-1 ) or the TRPC6 positive modulator C20 (n = 11; 18 µg kg-1 ). Similar experiments were conducted while the muscles were passively stretched (n = 8-12), a manoeuvre that isolated the mechanical component of the reflex. Blood pressure, tension, renal sympathetic nerve activity (RSNA) and blood flow were recorded. Of the DRG cells innervating the triceps surae muscles, 85% stained positive for the TRPC6 antigen, and 45% of those cells co-expressed neurofilament-200. Both TRPC6 antagonists decreased the reflex pressor responses to static contraction (-32 to -42%; P < 0.05) and to passive stretch (-35 to -52%; P < 0.05), whereas C20 increased these responses (55-65%; P < 0.05). In addition, BI-749327 decreased the peak and integrated RSNA responses to both static contraction (-39 to -43%; P < 0.05) and passive stretch (-56 to -62%; P < 0.05), whereas C20 increased the RSNA to passive stretch only. The onset latency of the decrease or increase in RSNA occurred within 2 s of the onset of the manoeuvres (P < 0.05). Collectively, our results show that TRPC6 plays a key role in evoking the mechanical component of the exercise pressor reflex. KEY POINTS: The exercise pressor reflex plays a key role in the sympathetic and haemodynamic responses to exercise. This reflex is composed of two components, namely the mechanoreflex and the metaboreflex. The receptors responsible for evoking the mechanoreflex are poorly documented. A good candidate for this function is the transient receptor potential canonical 6 (TRPC6) channel, which is activated by mechanical stimuli and expressed in dorsal root ganglia of rats. Using two TRPC6 antagonists and one positive modulator, we investigated the role played by TRPC6 in evoking the mechanoreflex in decerebrated rats. Blocking TRPC6 decreased the renal sympathetic and the pressor responses to both contraction and stretch, the latter being a manoeuvre that isolates the mechanoreflex. In contrast, the positive modulator increased the pressor reflex to contraction and stretch, in addition to the sympathetic response to stretch. Our results provide strong support for a role played by the TRPC6 channel in evoking the mechanoreflex.
RESUMO
Here, we report a bioluminescence resonance energy transfer (BRET) assay as a novel way to investigate the binding of unlabeled ligands to the human transient receptor potential mucolipin 1 (hTRPML1), a lysosomal ion channel involved in several genetic diseases and cancer progression. This novel BRET assay can be used to determine equilibrium and kinetic binding parameters of unlabeled compounds to hTRPML1 using intact human-derived cells, thus complementing the information obtained using functional assays based on ion channel activation. We expect this new BRET assay to expedite the identification and optimization of cell-permeable ligands that interact with hTRPML1 within the physiologically relevant environment of lysosomes.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Canais de Potencial de Receptor Transitório , Humanos , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Ligantes , Lisossomos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Autosomal dominant polycystic kidney disease is caused by mutations in PKD1 or PKD2 genes. The latter encodes polycystin-2 (PC2, also known as TRPP2), a member of the transient receptor potential ion channel family. Despite most pathogenic mutations in PKD2 being truncation variants, there are also many point mutations, which cause small changes in protein sequences but dramatic changes in the in vivo function of PC2. How these mutations affect PC2 ion channel function is largely unknown. In this study, we systematically tested the effects of 31 point mutations on the ion channel activity of a gain-of-function PC2 mutant, PC2_F604P, expressed in Xenopus oocytes. The results show that all mutations in the transmembrane domains and channel pore region, and most mutations in the extracellular tetragonal opening for polycystins domain, are critical for PC2_F604P channel function. In contrast, the other mutations in the tetragonal opening for polycystins domain and most mutations in the C-terminal tail cause mild or no effects on channel function as assessed in Xenopus oocytes. To understand the mechanism of these effects, we have discussed possible conformational consequences of these mutations based on the cryo-EM structures of PC2. The results help gain insight into the structure and function of the PC2 ion channel and the molecular mechanism of pathogenesis caused by these mutations.
Assuntos
Mutação com Ganho de Função , Mutação Puntual , Rim Policístico Autossômico Dominante , Canais de Cátion TRPP , Humanos , Microscopia Crioeletrônica , Oócitos/metabolismo , Mutação Puntual/genética , Rim Policístico Autossômico Dominante/genética , Relação Estrutura-Atividade , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Xenopus laevisRESUMO
Transmission is the first step for a microorganism to establish colonization in the respiratory tract and subsequent development of infectious disease. Streptococcus pneumoniae is a leading pathogen that colonizes the mucosal surfaces of the human upper respiratory tract and causes subsequent transmission and invasive infections especially in co-infection with influenza A virus. Host factors contributing to respiratory contagion are poorly understood. Transient receptor potential vanilloid (TRPV) channels have various roles in response to microoorganism. Inhibition of TRPV exacerbates invasive infection by Streptococcus pneumoniae, but it is unclear how TRPV channels influence pneumococcal transmission. Here, we describe the effect of inhibition of TRPV1 on pneumococcal transmission. We adopted a TRPV1-deficient infant mouse model of pneumococcal transmission during co-infection with influenza A virus. We also analyzed the expression of nasal mucin or pro-inflammatory cytokines. TRPV1 deficiency attenuated pneumococcal transmission and shedding during co-infection with influenza A virus. TRPV1 deficiency suppressed the expression of nasal mucin. In addition, there were increases in the expression of tumor necrosis factor-α and type I interferon, followed by the suppressed replication of influenza A virus in TRPV1-deficient mice. Inhibition of TRPV1 was shown to attenuate pneumococcal transmission by reducing shedding through the suppression of nasal mucin during co-infection with influenza A virus. Inhibition of TRPV1 suppressed nasal mucin by modulation of pro-inflammatory responses and regulation of replication of influenza A virus. TRPV1 could be a new target in preventive strategy against pneumococcal transmission.
RESUMO
In the adult brain, the water channel aquaporin-4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called "Orthogonal Arrays of Particles" (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS-PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time-related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca2+ transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca2+ dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development.
Assuntos
Astrócitos , Canais de Cátion TRPV , Astrócitos/metabolismo , Canais de Cátion TRPV/metabolismo , Aquaporina 4/metabolismo , Neuroglia/metabolismo , Encéfalo/metabolismoRESUMO
BACKGROUND: Temperature is an important environment factor that is critical to the survival and growth of crustaceans. However, the mechanisms by which crustaceans detect changes in temperature are still unclear. The transient receptor potential (TRP) channels are non-selective cation channels well known for properties in temperature sensation. However, comprehensive understandings on TRP channels as well as their temperature sensing functions are still lacking in crustaceans. RESULTS: In this study, a total of 26 TRP genes were identified in the swimming crab, Portunus trituberculatus, which can be classified into TRPA, TRPC, TRPP, TRPM, TRPML, TRPN and TRPV. Tissue expression analysis revealed a wide distribution of these TRP genes in P. trituberculatus, and antennules, neural tissues, and ovaries were the most commonly expressed tissues. To investigate the responsiveness of TRP genes to the temperature change, 18 TRPs were selected to detect their expression after high and low temperature stress. The results showed that 12 TRPs showed induced gene expression in both high and low temperature groups, while 3 were down-regulated in the low temperature group, and 3 showed no change in expression in either group. CONCLUSIONS: This study characterized the TRP family genes in P. trituberculatus, and explored their involvement in response to temperature stress. Our results will enhance overall understanding of crustacean TRP channels and their possible functions.
Assuntos
Braquiúros , Canais de Potencial de Receptor Transitório , Animais , Canais de Potencial de Receptor Transitório/genética , Braquiúros/genética , Temperatura , NataçãoRESUMO
Methylglyoxal (MGO), a highly reactive dicarbonyl metabolite of glucose primarily formed during the glycolytic pathway, is a precursor of advanced glycation end-products (AGEs). Recently, numerous studies have shown that MGO accumulation can cause pain and hyperalgesia. However, the mechanism through which MGO induces pain in the spinal dorsal horn remains unclear. The present study investigated the effect of MGO on spontaneous excitatory postsynaptic currents (sEPSC) in rat spinal dorsal horn neurons using blind whole-cell patch-clamp recording. Perfusion of MGO increased the frequency and amplitude of sEPSC in spinal horn neurons in a concentration-dependent manner. Additionally, MGO administration increased the number of miniature EPSC (mEPSC) in the presence of tetrodotoxin, a sodium channel blocker. However, 6-cyano-7-nitroqiunocaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist, blocked the enhancement of sEPSC by MGO. HC-030031, a TRP ankyrin-1 (TRPA1) antagonist, and capsazepine, a TRP vanilloid-1 (TRPV1) antagonist, inhibited the action of MGO. Notably, the effects of MGO were completely inhibited by HC-030031 and capsazepine. MGO generates reactive oxygen species (ROS) via AGEs. ROS also potentially induce pain via TRPA1 and TRPV1 in the spinal dorsal horn. Furthermore, we examined the effect of MGO in the presence of N-tert-butyl-α-phenylnitrone (PBN), a non-selective ROS scavenger, and found that the effect of MGO was completely inhibited. These results suggest that MGO increases spontaneous glutamate release from the presynaptic terminal to spinal dorsal horn neurons through TRPA1, TRPV1, and ROS and could enhance excitatory synaptic transmission.