ACTIVATION OF THE TRPV4 ION CHANNEL IS ENHANCED BY PHOSPHORYLATION

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Hueng-Chuen Fan^1,2, Xuming Zhang¹, Peter A McNaughton^1*

The TRPV4 ion channel shows substantial homology to TRPV1, the first member of the vanilloid subclass of TRP ion channels to be cloned. Like TRPV1, TRPV4 is activated by a wide range of stimuli, which amongst the possible physiological activators include membrane stretch caused by cell swelling or mechanical stress; warm temperatures above c. 27ºC; low pH; nitric oxide; and a variety of intracellular lipid messengers. The physiological role of TRPV4, and which of these possible activating stimuli is physiologically relevant, remains unknown. TRPV4 is highly expressed in renal nephron and in hypothalamus, and through its osmosensory properties may play a role in regulating body fluids (62;63), but is also expressed in bladder epithelium, where it may play a role in bladder voiding (64;65) and in sensory neurons, where roles both in detection of strong mechanical stimuli (66) and sensation of warm temperatures (67) have been proposed.

Tissue damage and inflammation cause the release of a range of pro-inflammatory mediators, with bradykinin and prostaglandins prominent among them. These mediators activate of intracellular signaling pathways and downstream kinases, amongst which PKA and PKC are known to be physiologically important (28). Both PKA and PKC enhance activation of TRPV1 (68). In the present paper we examined whether TRPV4 is modulated in a similar way to TRPV1 by phosphorylation by PKA and PKC. We used membrane stretch as a convenient activator of TRPV4, and we monitored activation of TRPV4 from the calcium influx when the channel opens. We carried out our experiments in a HEK293 cell expression system. We note that in the case of the related TRPV1 ion channel, studies in our lab and elsewhere have found that expression systems provide a highly reliable guide to the behaviour of the ion channel in its native environment (45; 69;70).

Activation of PKC by PMA, a potent and specific activator, substantially enhanced both the gating and the phosphorylation of TRPV4. The physiological pro-inflammatory mediator bradykinin, which activates G_q and hence PKC, also potentiated activation of TRPV4. The effect of PMA was inhibited by the broad-spectrum kinase inhibitor staurosporine, by the specific PKC inhibitor BIM I and by the inhibitor rottlerin, which has been proposed to be a specific inhibitor of PKCd (but see 58). These results show that PKC sensitizes TRPV4, as it does in the case of TRPV1. We indentified three phosporylation sites close together in the N-terminal domain, S162, T175, and S189, mutation of each of which partially inhibited the enhancement caused by PKC. Mutation of other candidate residues was without effect. Mutation of all three sites together completely abolished the effect of PKC activation without a significant effect on gating of TRPV4. Similar experiments on TRPV1 have also identified candidate PKC phosphorylation sites, but in different parts of the molecule: S502 located in the S2-S3 linker, close to the binding site for capsaicin, and S801 located in the C terminal domain (71;72).

Activation of PKA by application of the adenylate cyclase activator FSK also enhanced gating of TRPV4. The specific PKA inhibitor H89 abolished the effect of FSK, confirming that PKA is involved. A number of candidate phosphorylation sites was investigated, and amongst these a substantial inhibition of the enhancement, though not a complete abolition, was observed following mutation of a single site, S824, in the C-terminal domain.

Finally, by using a combination of co-expression and knock down approaches, AKAP79 was shown to play a vital role in tethering PKA and PKC to TRPV4 in order to modulate its gating. Functional studies using calcium imaging showed that AKAP79 overexpression enhanced sensitization of TRPV4 by FSK (Figure 4A and E) and PMA (Figure 4B and F), while downregulation of AKAP79 using siRNA inhibited sensitization. These functional studies were supported by studies of the effect of AKAP79 overexpression or downregulation on the phosphorylation of TRPV4, in which it was shown that overexpression of AKAP79 enhanced the phosphorylation induced by PMA (Figure 5C and D), while knockdown with siRNA against AKAP79 decreased the effect of PKC activation on phosphorylation (Figure 5C and D).

In summary, the gating of the TRPV4 ion channel by cell swelling is modulated by phosphorylation by the S/T kinases PKA and PKC in a manner reminiscent of TRPV1, though at sites located in quite different places on the protein. As is the case with TRPV1, AKAP79 orchestrates the action of PKC and PKA by tethering these kinases to TRPV4 so as to enhance the function and phosphorylation of the targeted ion channel. Manipulating this signaling integrator could be a promising target for the development of novel analgesics.