Activation of the TRPV4 Ion Channel Is Enhanced by Phosphorylation

Abstract

The TRPV4 (transient receptor potential vanilloid 4) ion channel, a member of the vanilloid subfamily of the transient receptor potential channels, is activated by membrane stretch, by non-noxious warm temperatures, and by a range of chemical activators. In the present study we examined the role of phosphorylation in modulating the activation of TRPV4. We expressed TRPV4 in HEK293 cells and activated the channel by cell swelling in a hypotonic solution. TRPV4 channel activation and serine phosphorylation were enhanced by exposure to the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate or by application of bradykinin, which activates PKC via a G-protein-coupled mechanism. The enhancement was inhibited by the PKC inhibitors staurosporine, bisindolylmaleimide I, and rottlerin or by mutation of the serine/threonine residues Ser 162 , Thr 175 , and Ser 189 . The adenylate cyclase activator forskolin also enhanced activation of TRPV4, and the enhancement was antagonized by the selective cyclic AMP-dependent protein kinase (PKA) inhibitor H89 or by mutation of serine residue Ser 824 . Sensitization of TRPV4 by both PKC and PKA depended on the scaffolding protein AKAP79, because channel activation and phosphorylation were enhanced by co-transfection of AKAP79 and were antagonized by removal of AKAP79 using small interfering RNA. We conclude that the serine/threonine kinases PKC and PKA enhance activation of the TRPV4 ion channel by phosphorylation at specific sites and that phosphorylation depends on assembly of PKC and PKA by AKAP79 into a signaling complex with TRPV4.TRPV4 was cloned from kidney, hypothalamus, and auditory epithelium and was given a number of names: OTRPC4 (Osm-9-like TRP channel 4) (1), VR-OAC (2), TRP12 (3), and VRL-2 (vanilloid receptor-like channel 2) (4). The gene for human TRPV4 is located on chromosome 12q23-q24.1 and has 15 exons, which code for a full-length protein with 871 amino acids. TRPV4 is a member of the transient receptor potential vanilloid subfamily of TRP 2 channels, and like other members of this subfamily, it is a polymodal receptor activated by a wide variety of stimuli. TRPV4 is strongly expressed in kidney and is activated by hypotonicity, which has led to the suggestion that TRPV4 is an osmosensor important in regulating body fluid levels (2, 5-9). However, TRPV4 is also activated by innocuous heat with a threshold of Ͼ27°C (6, 10, 11), by the phorbol ester 4␣-phorbol 12,13-didecanoate (12, 13), by low pH (14), by endocannabinoids and arachidonic acid metabolites (15, 16), by the active compound, bisandrographolide A, of Andrographis paniculata, a Chinese herbal plant (17), and by nitric oxide (18). TRPV4 is expressed in a broad range of tissues, including lung, spleen, kidney, testis, fat, brain, cochlea, skin, smooth muscle, liver, and vascular endothelium (1-3); in the lamina terminalis of the mouse brain; in neurons of the arched vascular organ of the lamina terminalis; and in the median preoptic area, the optic chiasm, neurons of the subfornical organ, t...

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

“…Considered together, these results suggest that in DRG neurons elastase-activated PAR 2 causes an adenylyl cyclase-and PKA-dependent activation of TRPV4. They are consistent with the known involvement of PKA in regulating TRPV4 (63), and our observation that cathepsin S also causes a PKA-mediated activation of TRPV4 in DRG neurons (31). PAR 2 coupling to TRPV4 has been observed in multiple systems with a variety of PAR 2 agonists.…”

Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

“…Although S ‐nitrosylation and activation of TRPV4 channels by NO cannot be ruled out, our results suggest that NO‐induced TRPV4 channel inhibition via GC‐PKG pathway predominates in intact PAs. Unlike PKG, protein kinase A and protein kinase C activation potentiates TRPV4 channel function via channel phosphorylation 69. These findings point to an interesting possibility that PKG phosphorylates the channel at a site that is different from protein kinase C or protein kinase A phosphorylation, and results in channel inhibition.…”

Discrete Control of TRPV4 Channel Function in the Distal Nephron by Protein Kinases A and C

“…However, the cGMP/PKG pathway has no direct inhibitory actions on TRPV4, but it acts on its heteromeric counterpart, TRPP2 (17). In contrast, experimental evidence in expression systems indicates that intracellular N and C termini of TRPV4 can be subjected to direct phosphorylation by PKC and PKA, resulting in augmentation of cellular responses to mechanical stress elicited by hypotonicity (19). However, it is unclear whether PKC and PKA play a role in regulation of TRPV4-mediated mechanosensitivity in the mammalian distal nephron.…”