The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na(+), K(+), Ca(2+) and Mg(2+)) flux across the cell membrane, and is implicated in cell-volume regulation, cell locomotion, muscle dystrophy and cardiac arrhythmias. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp. The oocyte was chosen because it expresses the prototypical MscCa (>or=10(7)MscCa/oocyte) that is preserved in cytoskeleton-deficient membrane vesicles. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (M(r) 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1). Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels.

This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca(2+) permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using a fast pressure clamp system. Although both TRPC1 and TRPC6 are highly expressed at the protein level, the amplitude of the mechano-sensitive current is not significantly altered by overexpression of these subunits. In conclusion, although several TRPC channel members, including TRPC1 and TRPC6, have been recently proposed to form MscCa in vertebrate cells, the functional expression of these TRPC subunits in heterologous systems remains problematic.

Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity.Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing.Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS.Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures.Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

Many animal cells release ATP into the extracellular medium, and often this release is mechanosensitive. However, the mechanisms underlying this release are not well understood. Using the luciferin-luciferase bioluminescent assay we demonstrate that a Xenopus oocyte releases ATP at a basal rate ϳ0.01 fmol/s, and gentle mechanical stimulation can increase this to 50 fmol/s. Brefeldin A, nocodazole, and progesterone-inducedmaturation block basal and mechanosensitive ATP release. These treatments share the common feature of disrupting the Golgi complex and vesicle trafficking to the cell surface and thereby block protein secretion and membrane protein insertion. We propose that ATP release occurs when protein transport vesicles enriched in ATP fuse with the plasma membrane. Collagenase, integrin-binding peptides, and cytochalasin D also block ATP release, indicating that extracellular, membrane and cytoskeletal elements are involved in the release process. Elevation of intracellular Ca 2؉ does not evoke ATP release but potentiates mechanosensitive ATP release. Our study indicates a novel mechanism of mechanotransduction that would allow cells to regulate membrane trafficking and protein transport/secretion in response to mechanical loading.Many, if not all, animal cells release ATP (or UTP) into the extracellular medium, and often this release is MS 1 (1-6). External ATP acts on ATP receptors that regulate diverse functions, including pain and touch sensation, smooth muscle contractility, synaptic transmission, platelet aggregation, epithelial fluid secretion, and endothelial release of vasorelaxants (6 -8). Furthermore, abnormalities in ATP release may contribute to specific human diseases, most notably cystic fibrosis (9). Although several mechanisms have been proposed to contribute to ATP release, including synaptic vesicular release and various membrane ion channels (10 -16), the mechanism of MS ATP release remains unknown.Our interest in MS ATP release was stimulated by the discovery of Nakamura and Strittmatter (1) that mechanical stimulation of the Xenopus oocyte evokes ATP release without causing an increase in membrane conductance. Here, we test the hypothesis that ATP release from the oocyte is mediated by the high rate (4,000 -16,000/s) of membrane fusion of vesicles involved in transporting proteins from the Golgi complex to the cell surface (17). This idea seemed plausible given the identification of a specific ATP transporter that concentrates ATP in the Golgi/ER lumen 50 -100-fold above that in the cytoplasm (18,19). To test the hypothesis we have examined the effects of BFA and other treatments that are known to disrupt membrane trafficking and thereby block protein secretion (20 -22).A key issue for any MS process is the pathway by which mechanical forces are transmitted to that process. For example, specific membrane channel proteins in bacteria and animal cells respond directly to tension developed in the lipid bilayer (23), while other MS processes may be activated by forces transmitted via elem...

NFκB/RelA triggers innate inflammation by binding to Bromodomain-Containing Protein 4 (BRD4), an atypical histone acetyltransferase (HAT). Although RelA·BRD4 HAT mediates acute neutrophilic inflammation, its role in chronic and functional airway remodeling is not known. We observed that BRD4 is required for TLR3 mediated mesenchymal transition, a cell-state change that is characteristic of remodeling. We therefore tested novel highly selective BRD4 inhibitors, ZL0420 and -0454, on chronic airway remodeling produced by repetitive TLR3 agonist challenges, and compared their efficacy with nonselective BET protein inhibitors, JQ1 and RVX208. We observed that ZL0420 and -0454 more potently reduced poly(I:C)-induced weight loss, fibrosis assessed by micro-CT and second harmonic generation microscopy; these measures correlated with collagen deposition observed in histopathology. Importantly the ZL inhibitors were more effective than that of nonselective BET inhibitors at equivalent doses. The ZL inhibitors had significant effects on lung physiology, reversing TLR3-associated airway hyper responsiveness (AHR) and increasing lung compliance in vivo. At the molecular level, ZL inhibitors reduced elaboration of the TGF β-induced growth program, preventing mucosal mesenchymal transition, disrupting BRD4 HAT activity, and complex formation with RelA. We also observed that ZL0454 treatment blocked poly(I:C)-associated expansion of the αSMA1+/COL1A+ myofibroblast population, and prevented myofibroblast transition in a co-culture system. We conclude that 1) BRD4 is a central effector of mesenchymal transition that results in paracrine activation of myofibroblasts, mechanistically linking innate inflammation to AHR and fibrosis, and 2) highly selective BRD4 inhibitors may be effective in reversing the effects of repetitive airway viral infections on innate inflammation-mediated remodeling.