A role for AQP5 in activation of TRPV4 by hypotonicity: concerted involvement of AQP5 and TRPV4 in regulation of cell volume recovery

Regulation of cell volume in response to changes in osmolarity is critical for cell function and survival. However, the molecular basis of osmosensation and regulation of cell volume are not clearly understood. We have examined the mechanism of regulatory volume decrease (RVD) in salivary gland cells and report a novel association between osmosensing TRPV4 (transient receptor potential vanalloid 4) and AQP5 (aquaporin 5), which is required for regulating water permeability and cell volume. Exposure of salivary gland cells and acini to hypotonicity elicited an increase in cell volume and activation of RVD. Hypotonicity also activated Ca2+ entry, which was required for subsequent RVD. Ca2+ entry was associated with a distinct nonselective cation current that was activated by 4alphaPDD and inhibited by ruthenium red, suggesting involvement of TRPV4. Consistent with this, endogenous TRPV4 was detected in cells and in the apical region of acini along AQP5. Importantly, acinar cells from mice lacking either TRPV4 or AQP5 displayed greatly reduced Ca2+ entry and loss of RVD in response to hypotonicity, although the extent of cell swelling was similar. Expression of N terminus-deleted AQP5 suppressed TRPV4 activation and RVD but not cell swelling. Furthermore, hypotonicity increased the association and surface expression of AQP5 and TRPV4. Both these effects and RVD were reduced by actin depolymerization. These data demonstrate that (i) activation of TRPV4 by hypotonicity depends on AQP5, not on cell swelling per se, and (ii) TRPV4 and AQP5 concertedly control regulatory volume decrease. These data suggest a potentially important role for TRPV4 in salivary gland function.     

Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells

TRPV4 is a non-selective cation channel with moderate calcium permeability, which is activated by exposure to hypotonicity. Such a stress induces regulatory volume decrease (RVD) behavior in human corneal epithelial cells (HCEC). We hypothesize that TRPV4 channel mediates RVD in HCEC. Immunohistochemistry revealed centrally and superficially concentrated TRPV4 localization in the corneal tissue. Immunocytochemical and fluorescence activated cell sorter (FACS) analyses identified TRPV4 membrane surface and cytosolic expression. RT-PCR and Western blot analyses identified TRPV4 gene and protein expression in HCEC, respectively. In addition, 4alpha-PDD or a 50% hypotonic medium induced up to threefold transient intracellular Ca(2+) ([Ca(2+)](i)) increases. Following TRPV4 siRNA HCEC transfection, its protein expression level declined by 64%, which abrogated these [Ca(2+)](i) transients. Similarly, exposure to either ruthenium red or Ca(2+)-free Ringer's solution also eliminated this response. In these transfected cells, RVD declined by 51% whereas in the non-transfected counterpart, ruthenium red and Ca(2+)-free solution inhibited RVD by 54 and 64%, respectively. In contrast, capsazepine, a TRPV1 antagonist, failed to suppress [Ca(2+)](i) transients and RVD. TRPV4 activation contributes to RVD since declines in TRPV4 expression and activity are associated with suppression of this response. In conclusion, there is TRPV4 functional expression in HCEC.     

Aquaporin-2 regulates cell volume recovery via tropomyosin

Cell volume regulation is particularly important for kidney collecting duct cells. These cells are the site of water reabsorption regulated by vasopressin and aquaporin-2 (AQP2) trafficking to the apical membrane, and subject to changes in osmolality. Here, we examined the role of AQP2 in regulatory volume decrease (RVD), which is a cellular defensive process against hypotonic stress. Stable expression of AQP2 increases RVD in MDCK cells and its phosphorylation levels decrease during the RVD process. We then examined the involvement of AQP2 phosphorylation at serine 256 and serine 261 in RVD using cells stably expressing the phosphorylation mutants. Both S256A- and S256D-AQP2 decrease RVD compared to wild type (WT)-AQP2 although only S256A mutation decreases the initial osmotic swelling, indicating that AQP2-enhanced RVD is independent of osmotic swelling induced by the water permeability of AQP2. S261A and S261D mutations do not induce changes compared with WT-AQP2. These findings indicate that switching between phosphorylation and dephosphorylation at S256 is important for RVD. We previously reported that AQP2 interacts with tropomyosin 5b (TM5b), which regulates actin stability. AQP2 interactions with TM5b are rapidly increased by hypotonicity and then decreased, which are consistent with AQP2 phosphorylation levels. Knockdown and overexpression of TM5b show its essential role in WT-AQP2-enhanced RVD. RVD in S256A- and S256D-AQP2-expressing cells is not changed by TM5b knockdown or overexpression. The present study shows that AQP2 regulates RVD via TM5b and switching between phosphorylation and dephosphorylation at S256 in AQP2 is critical for this process.     

Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia

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 alpha-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4 alpha-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.     

Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration

In the journey from the male to female reproductive tract, mammalian sperm experience a natural osmotic decrease (e.g., in mouse, from ~415 mOsm in the cauda epididymis to ~310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation, meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however, no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here, we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling, characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet, forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct, resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the "trade-off" between sperm motility and cell swelling upon physiological hypotonicity, thereby optimizing postcopulatory sperm behavior.     

PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells

Transient receptor potential vanilloid channel 4 (TRPV4) is a polymodally activated nonselective cationic channel implicated in the regulation of vasodilation and hypertension. We and others have recently shown that cyclic stretch and shear stress activate TRPV4-mediated calcium influx in endothelial cells (EC). In addition to the mechanical forces, acetylcholine (ACh) was shown to activate TRPV4-mediated calcium influx in endothelial cells, which is important for nitric oxide-dependent vasodilation. However, the molecular mechanism through which ACh activates TRPV4 is not known. Here, we show that ACh-induced calcium influx and endothelial nitric oxide synthase (eNOS) phosphorylation but not calcium release from intracellular stores is inhibited by a specific TRPV4 antagonist, AB-159908. Importantly, activation of store-operated calcium influx was not altered in the TRPV4 null EC, suggesting that TRPV4-dependent calcium influx is mediated through a receptor-operated pathway. Furthermore, we found that ACh treatment activated protein kinase C (PKC) α, and inhibition of PKCα activity by the specific inhibitor Go-6976, or expression of a kinase-dead mutant of PKCα but not PKCε or downregulation of PKCα expression by chronic 12-O-tetradecanoylphorbol-13-acetate treatment, completely abolished ACh-induced calcium influx. Finally, we found that ACh-induced vasodilation was inhibited by the PKCα inhibitor Go-6976 in small mesenteric arteries from wild-type mice, but not in TRPV4 null mice. Taken together, these findings demonstrate, for the first time, that a specific isoform of PKC, PKCα, mediates agonist-induced receptor-mediated TRPV4 activation in endothelial cells.     

Maxi K+ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line

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.     

Roles of Aquaporin-3 Water Channels in Volume-Regulatory Water Flow in a Human Epithelial Cell Line

Membrane water transport is an essential event not only in the osmotic cell volume change but also in the subsequent cell volume regulation. Here we investigated the route of water transport involved in the regulatory volume decrease (RVD) that occurs after osmotic swelling in human epithelial Intestine 407 cells. The diffusion water permeability coefficient (Pd) measured by NMR under isotonic conditions was much smaller than the osmotic water permeability coefficient (Pf) measured under an osmotic gradient. Temperature dependence of Pf showed the Arrhenius activation energy (Ea) of a low value (1.6 kcal/mol). These results indicate an involvement of a facilitated diffusion mechanism in osmotic water transport. A mercurial water channel blocker (HgCl₂) diminished the Pf value. A non-mercurial sulfhydryl reagent (MMTS) was also effective. These blockers of water channels suppressed the RVD. RT-PCR and immunocytochemistry demonstrated predominant expression of AQP3 water channel in this cell line. Downregulation of AQP3 expression induced by treatment with antisense oligodeoxynucleotides was found to suppress the RVD response. Thus, it is concluded that AQP3 water channels serve as an essential pathway for volume-regulatory water transport in, human epithelial cells.     

IP3 sensitizes TRPV4 channel to the mechano- and osmotransducing messenger 5'-6'-epoxyeicosatrienoic acid

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.     

Aquaporin-2 and Na+/H+ exchanger isoform 1 modulate the efficiency of renal cell migration

Aquaporin-2 (AQP2) promotes renal cell migration by the modulation of integrin β1 trafficking and the turnover of focal adhesions. The aim of this study was to investigate whether AQP2 also works in cooperation with Na⁺/H⁺ exchanger isoform 1 (NHE1), another well-known protein involved in the regulation of cell migration. Our results showed that the lamellipodia of AQP2-expressing cells exhibit significantly smaller volumes and areas of focal adhesions and more alkaline intracellular pH due to increased NHE1 activity than AQP2-null cells. The blockage of AQP2, or its physically-associated calcium channel TRPV4, significantly reduced lamellipodia NHE1 activity. NHE1 blockage significantly reduced the rate of cell migration, the number of lamellipodia, and the assembly of F-actin only in AQP2-expressing cells. Our data suggest that AQP2 modulates the activity of NHE1 through its calcium channel partner TRPV4, thereby determining pH-dependent actin polymerization, providing mechanical stability to delineate lamellipodia structure and defining the efficiency of cell migration.     

Hypotonicity-induced TRPV4 function in renal collecting duct cells: modulation by progressive cross-talk with Ca2+-activated K+ channels

The mouse cortical collecting duct (CCD) M-1 cells were grown to confluency on coverslips to assess the interaction between TRPV4 and Ca(2+)-activated K(+) channels. Immunocytochemistry demonstrated strong expression of TRPV4, along with the CCD marker, aquaporin-2, and the Ca(2+)-activated K(+) channels, the small conductance SK3 (K(Ca)2.3) channel and large conductance BKα channel (K(Ca)1.1). TRPV4 overexpression studies demonstrated little physical dependency of the K(+) channels on TRPV4. However, activation of TRPV4 by hypotonic swelling (or GSK1016790A, a selective agonist) or inhibition by the selective antagonist, HC-067047, demonstrated a strong dependency of SK3 and BK-α activation on TRPV4-mediated Ca(2+) influx. Selective inhibition of BK-α channel (Iberiotoxin) or SK3 channel (apamin), thereby depolarizing the cells, further revealed a significant dependency of TRPV4-mediated Ca(2+) influx on activation of both K(+) channels. It is concluded that a synergistic cross-talk exists between the TRPV4 channel and SK3 and BK-α channels to provide a tight functional regulation between the channel groups. This cross-talk may be progressive in nature where the initial TRPV4-mediated Ca(2+) influx would first activate the highly Ca(2+)-sensitive SK3 channel which, in turn, would lead to enhanced Ca(2+) influx and activation of the less Ca(2+)-sensitive BK channel.     

IP3 receptor binds to and sensitizes TRPV4 channel to osmotic stimuli via a calmodulin-binding site

Activation of the non-selective cation channel TRPV4 by mechanical and osmotic stimuli requires the involvement of phospholipase A2 and the subsequent production of the arachidonic acid metabolites, epoxieicosatrienoic acids (EET). Previous studies have shown that inositol trisphosphate (IP3) sensitizes TRPV4 to mechanical, osmotic, and direct EET stimulation. We now search for the IP3 receptor-binding site on TRPV4 and its relevance to IP3-mediated sensitization. Three putative sites involved in protein-protein interactions were evaluated: a proline-rich domain (PRD), a calmodulin (CaM)-binding site, and the last four amino acids (DAPL) that show a PDZ-binding motif-like. TRPV4-DeltaCaM-(Delta812-831) channels preserved activation by hypotonicity, 4alpha-phorbol 12,13-didecanoate, and EET but lost their physical interaction with IP3 receptor 3 and IP3-mediated sensitization. Deletion of a PDZ-binding motif-like (TRPV4-DeltaDAPL) did not affect channel activity or IP3-mediated sensitization, whereas TRPV4-DeltaPRD-(Delta132-144) resulted in loss of channel function despite correct trafficking. We conclude that IP3-mediated sensitization requires IP3 receptor binding to a TRPV4 C-terminal domain that overlaps with a previously described calmodulin-binding site.     

Regulation of a transient receptor potential (TRP) channel by tyrosine phosphorylation. SRC family kinase-dependent tyrosine phosphorylation of TRPV4 on TYR-253 mediates its response to hypotonic stress

The recently identified transient receptor potential (TRP) channel family member, TRPV4 (formerly known as OTRPC4, VR-OAC, TRP12, and VRL-2) is activated by hypotonicity. It is highly expressed in the kidney as well as blood-brain barrier-deficient hypothalamic nuclei responsible for systemic osmosensing. Apart from its gating by hypotonicity, little is known about TRPV4 regulation. We observed that hypotonic stress resulted in rapid tyrosine phosphorylation of TRPV4 in a heterologous expression model and in native murine distal convoluted tubule cells in culture. This tyrosine phosphorylation was sensitive to the inhibitor of Src family tyrosine kinases, PP1, in a dose-dependent fashion. TRPV4 associated with Src family kinases by co-immunoprecipitation studies and confocal immunofluorescence microscopy, and this interaction required an intact Src family kinase SH2 domain. One of these kinases, Lyn, was activated by hypotonic stress and phosphorylated TRPV4 in an immune complex kinase assay and an in vitro kinase assay using recombinant Lyn and TRPV4. Transfection of wild-type Lyn dramatically potentiated hypotonicity-dependent TRPV4 tyrosine phosphorylation whereas dominant negative-acting Lyn modestly inhibited it. Through mutagenesis studies, the site of tonicity-dependent tyrosine phosphorylation was mapped to Tyr-253, which is conserved across all species from which TRPV4 has been cloned. Importantly, point mutation of Tyr-253 abolished hypotonicity-dependent channel activity. In aggregate, these data indicate that hypotonic stress results in Src family tyrosine kinase-dependent tyrosine phosphorylation of the tonicity sensor TRPV4 at residue Tyr-253 and that this residue is essential for channel function in this context. This is the first example of direct regulation of TRP channel function through tyrosine phosphorylation.     

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.     

Aquaporin 2‐increased renal cell proliferation is associated with cell volume regulation

We have previously demonstrated that in renal cortical collecting duct cells (RCCD₁) the expression of the water channel Aquaporin 2 (AQP2) raises the rate of cell proliferation. In this study, we investigated the mechanisms involved in this process, focusing on the putative link between AQP2 expression, cell volume changes, and regulatory volume decrease activity (RVD). Two renal cell lines were used: WT‐RCCD₁ (not expressing aquaporins) and AQP2‐RCCD₁ (transfected with AQP2). Our results showed that when most RCCD₁ cells are in the G₁‐phase (unsynchronized), the blockage of barium‐sensitive K⁺ channels implicated in rapid RVD inhibits cell proliferation only in AQP2‐RCCD₁ cells. Though cells in the S‐phase (synchronized) had a remarkable increase in size, this enhancement was higher and was accompanied by a significant down‐regulation in the rapid RVD response only in AQP2‐RCCD₁ cells. This decrease in the RVD activity did not correlate with changes in AQP2 function or expression, demonstrating that AQP2—besides increasing water permeability—would play some other role. These observations together with evidence implying a cell‐sizing mechanism that shortens the cell cycle of large cells, let us to propose that during nutrient uptake, in early G₁, volume tends to increase but it may be efficiently regulated by an AQP2‐dependent mechanism, inducing the rapid activation of RVD channels. This mechanism would be down‐regulated when volume needs to be increased in order to proceed into the S‐phase. Therefore, during cell cycle, a coordinated modulation of the RVD activity may contribute to accelerate proliferation of cells expressing AQP2. J. Cell. Biochem. 113: 3721–3729, 2012. © 2012 Wiley Periodicals, Inc.     

Serotonin-induced activation of TRPV4-like current in rat intrapulmonary arterial smooth muscle cells

In the present study, we investigated the implication of transient receptor potential vanilloid (TRPV)-related channels in the 5-hydroxytryptamine (5-HT)-induced both intracellular calcium response and mitogenic effect in rat pulmonary arterial smooth muscle cells (PASMC). Using microspectrofluorimetry (indo-1 as Ca(2+) fluorescent probe) and the patch-clamp technique (in whole-cell configuration), we found that 5-HT (10 microM) induced a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter was partly blocked by an inhibitor of cytochrome P450 epoxygenase (17-ODYA) and insensitive to cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and CDC), suggesting the involvement of arachidonic acid metabolization by cytochrome P450 epoxygenase. This calcium influx was also sensitive to Ni(2+) and to ruthenium red, a TRPV channel blocker, and mimicked by 4alpha-phorbol-12,13-didecanoate (4alpha-PDD), a TRPV4 channel agonist. In patched PASMC, 5-HT and 4alpha-PDD-activated TRPV4-like ruthenium red sensitive currents with typical characteristics. Furthermore, 5-HT induced a ruthenium red sensitive increase in BrdU incorporation levels in PASMC. The present study provides evidence that 5-HT activates a TRPV4-like current, potentially involved in PASMC proliferation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension.     

Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways

The TRPV4 calcium-permeable channel was cloned from mouse kidney M-1 cells, and the effect of temperature modulation on channel gating/activation by physical and chemical signals was evaluated. A TRPV4 cDNA construct with a C-terminal V5 epitope was stably transfected into human embryonic kidney (HEK) 293 and Chinese hamster ovary cells resulting in high levels of expression at the plasma membrane. Channel activation was assessed from changes in calcium influx (fura-2 fluorescence measurements) or whole cell currents (patch clamp analysis). At room temperature (22-24 degrees C), exposure of TRPV4-transfected cells to hypotonic medium (225 mOsm/liter) or a non-protein kinase C (PKC)-activating phorbol ester derivative, 4alpha-phorbol 12,13-decanoate (100 nm), induces modest channel activation, whereas phorbol 12-myristate 13-acetate (100 nm), a PKC-activating phorbol ester, and shear stress (3-20 dyne/cm2) had minimal or no effect on channel activation. In contrast, at elevated temperatures (37 degrees C) the channel was rapidly activated by all stimuli. Inhibition of PKC by calphostin C (50 nm) or staurosporine (500 nm) abolished phorbol 12-myristate 13-acetate-induced activation of the channel without affecting the response to other stimuli. Ruthenium red (1 microm) effectively blocked the channel activity by all stimuli. It is concluded that temperature is a critical modulator of TRPV4 channel gating, leading to activation of the channel by a diverse range of microenvironmental chemical and physical signals utilizing a least two transduction pathways, one PKC-dependent and one PKC-independent. The convergence of multiple signals and transduction pathways on the same channel indicate that the channel functions as a molecular integrator of microenvironmental chemical and physical signals.     

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle

Transient receptor potential vanilloid 4 (TRPV4) channels are Ca²⁺-permeable, nonselective cation channels expressed in multiple tissues, including smooth muscle. Although TRPV4 channels play a key role in regulating vascular tone, the mechanisms controlling Ca²⁺ influx through these channels in arterial myocytes are poorly understood. Here, we tested the hypothesis that in arterial myocytes the anchoring protein AKAP150 and protein kinase C (PKC) play a critical role in the regulation of TRPV4 channels during angiotensin II (AngII) signaling. Super-resolution imaging revealed that TRPV4 channels are gathered into puncta of variable sizes along the sarcolemma of arterial myocytes. Recordings of Ca²⁺ entry via single TRPV4 channels (“TRPV4 sparklets”) suggested that basal TRPV4 sparklet activity was low. However, Ca²⁺ entry during elementary TRPV4 sparklets was ∼100-fold greater than that during L-type Ca_V1.2 channel sparklets. Application of the TRPV4 channel agonist GSK1016790A or the vasoconstrictor AngII increased the activity of TRPV4 sparklets in specific regions of the cells. PKC and AKAP150 were required for AngII-induced increases in TRPV4 sparklet activity. AKAP150 and TRPV4 channel interactions were dynamic; activation of AngII signaling increased the proximity of AKAP150 and TRPV4 puncta in arterial myocytes. Furthermore, local stimulation of diacylglycerol and PKC signaling by laser activation of a light-sensitive G_q-coupled receptor (opto-α₁AR) resulted in TRPV4-mediated Ca²⁺ influx. We propose that AKAP150, PKC, and TRPV4 channels form dynamic subcellular signaling domains that control Ca²⁺ influx into arterial myocytes.     

Role of Calcium in Volume-Activated Chloride Currents in a Mouse Cholangiocyte Cell Line

Volume-activated Cl(-) channels (VACCs) play vital roles in many cells including cholangiocytes. Previously, we characterized the VACCs in mouse cholangiocytes. Since calcium plays an important role in VACC regulation in many cells, we have studied the effect of calcium modulation on the regulatory volume decrease (RVD) and VACC currents in mouse bile duct cells (MBDCs). Cell volume measurements were assessed by a Coulter counter with cell sizer, and conventional whole-cell patch-clamp techniques were used to study the role of calcium on RVD and VACC currents. Cell volume study indicated that MBDCs exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) but not by removal of extracellular calcium. During hypotonic challenge, MBDCs exhibited an outwardly rectified current, which was significantly inhibited by administration of classical chloride channel inhibitors such as NPPB and tamoxifen. Chelation of the intracellular calcium with BAPTA-AM or removal of extracellular calcium and calcium channel blocker had no significant effect on VACC currents during hypotonic challenge. In addition to VACC, MBDC had a calcium-activated chloride channel, which was inhibited by NPPB. The present study is the first to systemically study the role of calcium on the VACC and RVD in mouse cholangiocytes and demonstrates that a certain level of intracellular calcium is necessary for RVD but the activation of VACC during RVD does not require calcium. These findings suggest that calcium does not have a direct regulatory role on VACC but has a permissive role on RVD in cholangiocytes.     

Cyclic adenosine monophosphate-dependent activation of transient receptor potential vanilloid 4 (TRPV4) channels in osteoblast-like MG-63 cells

Parathyroid hormone (PTH) directly interacts with bone remodeling osteoblasts and osteocytes expressing the G-protein coupled receptor PTH receptor 1 (PTH1R), and its osteoanabolic effects mostly involve the cAMP/PKA signaling cascade. Considering that PTH-dependent calcium entry in rat enterocytes is reproduced by the adenylate cyclase agonist forskolin or by cAMP analogues, possible involvement of calcium as a second messenger in PTH-dependent cAMP signaling was investigated in MG-63 cells. First, Ca²⁺ influx was confirmed in Fluo3-loaded MG-63 cells treated with a cell-permeable cAMP analog. Second, PTH (1–34) and forskolin promoted calcium influxes that were completely abrogated by the PKA inhibitor H-89. Ca²⁺ entry was not reproduced when PTH (1–34) was combined with the PKC-activating competitor PTH (3–34). Vanilloid transient potential (TRPV) channel inhibitor Ruthenium Red, but not a voltage-dependent calcium channel (VDCC) inhibitor nifedipine, efficiently stunted Ca²⁺ entry, and comparable abrogation was reproduced in cells treated with TRPV4-selective inhibitor RN-1734 or transfected with TRPV4-specific siRNA. Interestingly, PTH-driven Ca²⁺ through TRPV4 significantly inhibited MG63 cell migration through a mechanism requiring extracellular Ca²⁺. In contrast, the inhibitory effects of forskolin on migration were refractory to TRPV4 silencing or to RN-1734. Altogether, our results indicate that single treatment with PTH (1–34) promotes extracellular calcium entry through TRPV4 channels in MG-63 cells through a cAMP/PKA-dependent mechanism, and that this influx affects cell migration.