Sensitisation of TRPV4 by PAR₂ is independent of intracellular calcium signalling and can be mediated by the biased agonist neutrophil elastase

Proteolytic activation of protease-activated receptor 2 (PAR₂) may represent a major mechanism of regulating the transient receptor potential vanilloid 4 (TRPV4) non-selective cation channel in pathophysiological conditions associated with protease activation (e.g. during inflammation). To provide electrophysiological evidence for PAR₂-mediated TRPV4 regulation, we characterised the properties of human TRPV4 heterologously expressed in Xenopus laevis oocytes in the presence and absence of co-expressed human PAR₂. In outside-out patches from TRPV4 expressing oocytes, we detected single-channel activity typical for TRPV4 with a single-channel conductance of about 100 pS for outward and 55 pS for inward currents. The synthetic TRPV4 activator GSK1016790A stimulated TRPV4 mainly by converting previously silent channels into active channels with an open probability of nearly one. In oocytes co-expressing TRPV4 and PAR₂, PAR₂ activation by trypsin or by specific PAR₂ agonist SLIGRL-NH₂ potentiated the GSK1016790A-stimulated TRPV4 whole-cell currents several fold, indicative of channel sensitisation. Pre-incubation of oocytes with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-AM did not reduce the stimulatory effect of PAR₂ activation on TRPV4, which indicates that the effect is independent of intracellular calcium signalling. Neutrophil elastase, a biased agonist of PAR₂ that does not induce intracellular calcium signalling, also caused a PAR₂-dependent sensitisation of TRPV4. The Rho-kinase inhibitor Y27362 abolished elastase-stimulated sensitisation of TRPV4, which indicates that Rho-kinase signalling plays a critical role in PAR₂-mediated TRPV4 sensitisation by the biased agonist neutrophil elastase. During acute inflammation, neutrophil elastase may sensitise TRPV4 by a mechanism involving biased agonism of PAR₂ and activation of Rho-kinase.