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.
Highlights
TRPV4 is an osmosensor important in regulating body fluid levels [2, 5,6,7,8,9]
Important intracellular signaling molecules contributing to inflammatory hyperalgesia include protein kinase C (PKC) [21, 22] and cyclic AMP-dependent protein kinase (PKA) [23]
The present study shows that PKC and PKA activation can sensitize TRPV4 to mechanical stimuli, identifies the relevant phosphorylation sites, and shows that the scaffolding protein AKAP79 plays a critical role in sensitization of TRPV4
Summary
Cell Culture and Transfection—HEK293 cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 g/ml streptomycin, and 20 mM L-glutamine. The siRNA against AKAP79 was constructed in a plasmid co-expressing green fluorescent protein to facilitate identification of successfully transfected cells, as previously described [35]. Following treatment as specified in figure legends, transfected cells were solubilized in lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 2 mM EGTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 10% protease inhibitors (Roche Applied Science), and 1ϫ phosphatase inhibitor (Sigma)), the cell lysates were centrifuged at 12,000 rpm for 10 min, and cleared supernatant was mixed with 1 l mouse anti-V5 to precipitate TRPV4 and 30 l of protein A-agarose (Santa Cruz) and was incubated for 3 h. All of the blots shown in the figures are typical of at least three similar results
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