Autoregulation of the ciliary beat frequency (CBF) has been proposed as the mechanism used by epithelial ciliated cells to maintain the CBF and prevent the collapse of mucociliary transport under conditions of varying mucus viscosity. Despite the relevance of this regulatory response to the pathophysiology of airways and reproductive tract, the underlying cellular and molecular aspects remain unknown. Hamster oviductal ciliated cells express the transient receptor potential vanilloid 4 (TRPV4) channel, which is activated by increased viscous load involving a phospholipase A2–dependent pathway. TRPV4-transfected HeLa cells also increased their cationic currents in response to high viscous load. This mechanical activation is prevented in native ciliated cells loaded with a TRPV4 antibody. Application of the TRPV4 synthetic ligand 4α-phorbol 12,13-didecanoate increased cationic currents, intracellular Ca2+, and the CBF in the absence of a viscous load. Therefore, TRPV4 emerges as a candidate to participate in the coupling of fluid viscosity changes to the generation of the Ca2+ signal required for the autoregulation of CBF.
The TRPV4 cation channel exhibits a topology consisting of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N-and C-tails, the former containing at least three ankyrin domains. Functional transient receptor potential (TRP) channels are supposed to result following the assembly of four subunits. However, the rules governing subunit assembly and protein domains implied in this process are only starting to emerge. The ankyrin, TM, and the C-tail domains have been identified as important determinants of the oligomerization process. We now describe the maturation and oligomerization of five splice variants of the TRPV4 channel. The already known TRPV4-A and TRPV4-B (⌬384 -444) variants and the new TRPV4-C (⌬237-284), TRPV4-D (⌬27-61), and TRPV4-E (⌬237-284 and ⌬384 -444) variants. All alternative spliced variants involved deletions in the cytoplasmic N-terminal region, affecting (except for TRPV4-D) the ankyrin domains. Subcellular localization, fluorescence resonance energy transfer, co-immunoprecipitation, glycosylation profile, and functional analysis of these variants permitted us to group them into two classes: group I (TRPV4-A and TRPV4-D) and group II (TRPV4-B, TRPV4-C, and TRPV4-E). Group I, unlike group II variants, were correctly processed, homo-and heteromultimerized in the endoplasmic reticulum, and were targeted to the plasma membrane where they responded to typical TRPV4 stimuli. Our results suggest that: 1) TRPV4 biogenesis involves core glycosylation and oligomerization in the endoplasmic reticulum followed by transfer to the Golgi apparatus for subsequent maturation; 2) ankyrin domains are necessary for oligomerization of TRPV4; and 3) lack of TRPV4 oligomerization determines its accumulation in the endoplasmic reticulum.The non-selective cation channel TRPV4 is a member of the transient receptor potential (TRP) 3 family of channels (1). TRPV4 shows multiple modes of activation and regulatory sites, enabling it to respond to various stimuli, including osmotic cell swelling (2-5), mechanical stress (6 -8), heat (9), acidic pH (7), endogenous ligands (10), high viscous solutions (11), and synthetic agonists such as 4␣-phorbol 12,13-didecanoate (4␣-PDD) (12). TRPV4 mRNA is expressed in a broad range of tissues (13), although functional tests have only been carried out in a few: endothelial (14), epithelial (5,11,15), smooth muscle (16), keratinocytes (17), and DRG neurons (18). An alternative splice variant lacking the seventh exon has also been cloned from human aortic endothelial cells (19), although this variant was not further investigated because of its unresponsiveness to hypotonic stimuli.The general topology of a TRP subunit consists of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N-and C-terminal regions of variable length, the former containing multiple ankyrin (ANK) repeats in the TRPC, TRPA, TRPN, and TRPV subfamilies (1). ANK repeats are modular protein interaction domains, ...
Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.
The vertebrate transient receptor potential cationic channel TRPV4 has been proposed as an osmo-and mechanosensor channel. Studies using knock-out animal models have further emphasized the relevance of the TRPV4 channel in the maintenance of the internal osmotic equilibrium and mechanosensation. However, at the cellular level, there is still one important question to answer: does the TRPV4 channel generate the Ca 2؉ signal in those cells undergoing a Ca 2؉ -dependent regulatory volume decrease (RVD) response? RVD in human airway epithelia requires the generation of a Ca 2؉ signal to activate Ca 2؉ -dependent K ؉ channels. The RVD response is lost in airway epithelia affected with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator channel. We have previously shown that the defective RVD in CF epithelia is linked to the lack of swelling-dependent activation of Ca 2؉ -dependent K ؉ channels. In the present study, we show the expression of TRPV4 in normal human airway epithelia, where it functions as the Ca 2؉ entry pathway that triggers the RVD response after hypotonic stress, as demonstrated by TRPV4 antisense experiments. However, cell swelling failed to trigger Ca 2؉ entry via TRPV4 channels in CF airway epithelia, although the channel's response to a specific synthetic activator, 4␣-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4␣-phorbol 12,13-didecanoate. Together, these results suggest that defective RVD in CF airway epithelia might be caused by the absence of a TRPV4-mediated Ca 2؉ signal and the subsequent activation of Ca 2؉ -dependent K ؉ channels.
Nonstandard abbreviations used: large conductance, Ca 2+ -dependent K + (BK); conductance (G); diastolic blood pressure (DBP); odds ratio (OR); open probability (PO).
Mechanical and osmotic sensitivity of the transient receptor potential vanilloid 4 (TRPV4) channel depends on phospholipase A2 (PLA2) activation and the subsequent production of the arachidonic acid metabolites, epoxyeicosatrienoic acid (EET). We show that both high viscous loading and hypotonicity stimuli in native ciliated epithelial cells use PLA2–EET as the primary pathway to activate TRPV4. Under conditions of low PLA2 activation, both also use extracellular ATP-mediated activation of phospholipase C (PLC)–inositol trisphosphate (IP3) signaling to support TRPV4 gating. IP3, without being an agonist itself, sensitizes TRPV4 to EET in epithelial ciliated cells and cells heterologously expressing TRPV4, an effect inhibited by the IP3 receptor antagonist xestospongin C. Coimmunoprecipitation assays indicated a physical interaction between TRPV4 and IP3 receptor 3. Collectively, our study suggests a functional coupling between plasma membrane TRPV4 channels and intracellular store Ca2+ channels required to initiate and maintain the oscillatory Ca2+ signal triggered by high viscosity and hypotonic stimuli that do not reach a threshold level of PLA2 activation.
The cell regulatory volume decrease (RVD) response triggered by hypotonic solutions is mainly achieved by the coordinated activity of Cl- and K+ channels. We now describe the molecular nature of the K(+) channels involved in the RVD response of the human bronchial epithelial (HBE) cell line 16HBE14o-. These cells, under isotonic conditions, present a K+ current consistent with the activity of maxi K+ channels, confirmed by RT-PCR and Western blot. Single-channel and whole cell maxi K+ currents were readily and reversibly activated following the exposure of HBE cells to a 28% hypotonic solution. Both maxi K+ current activation and RVD response showed calcium dependency, inhibition by TEA, Ba2+, iberiotoxin, and the cationic channel blocker Gd3+ but were insensitive to clofilium, clotrimazole, and apamin. The presence of the recently cloned swelling-activated, Gd3+-sensitive cation channels (TRPV4, also known as OTRPC4, TRP12, or VR-OAC) was detected by RT-PCR in HBE cells. This channel, TRPV4, which senses changes in volume, might provide the pathway for Ca2+ influx under hypotonic solutions and, consequently, for the activation of maxi K+ channels.
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