Modulation of the thermo- and hypotonicity-sensitive ion channel TRPV4

• Main Author: Fan, H.-C.
• Published: University of Cambridge 2008
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598933

In this thesis, the role of transient receptor potential vanilloid 4 (TRPV4) in mechanical hyperalgesia induced by inflammatory mediators and second-messenger pathways was investigated. The hypotonicity-activated [Ca²⁺]_I increase in HEK293 cells transiently expressing TRPV4 was greatly enhanced after exposure to the PKC activator phorbol 12-myristate 13-acetate (PMA). The sensitization was significantly reduced in the presence of kinase inhibitors, including staurosporine, BIM, and rottlerin, suggesting that a PKC-mediated phosphorylation was involved, and could be PKCδ mediated. The sensitization induced by PMA was also significantly reduced by the mutation S162A/T175A/S189A. The phosphorylation level of TRPV4 was greatly enhanced with PMA treatment, and was significantly decreased by the application of the PKCδ-specific inhibitor rottlerin or with the mutant S162A/T175A/S189A. These results suggest that sensitization was mediated by phosphorylation by PKCδ of S162, T175 and S189. The hypotonicity-activated [Ca²⁺]_i increase mediated by TRPV4 was enhanced after exposure to the cAMP activator forskolin (FSK). The sensitization was prevented in the presence of the selective PKA inhibitor H89. The sensitization induced by FSK was significantly reduced by the mutant S824A. The A kinase anchoring protein (AKAP) family is known to assemble a wide range of kinases, including PKA and PKC, into signalling complexes with appropriate targets. Functional studies using calcium imaging showed that AKAP79 overexpression enhanced sensitization of TRPV4 by FSK and PMA, while downregulation of AKAP79 using siRNA inhibited sensitization. TRPV4 coexpression with AKAP79 enhanced the phosphorylation induced by PMA, and the phosphorylation level significantly decreased in cells coexpressing TRPV4 and siRNA AKAP79. AKAP79 may be a critical component converging the effects of PKC and PKA on TRPV4.