Mechanisms of g-protein coupled receptor-dependent opening of transient receptor potential vanilloid-4

Darby, W 2019, Mechanisms of g-protein coupled receptor-dependent opening of transient receptor potential vanilloid-4, Doctor of Philosophy (PhD), Health and Biomedical Sciences, RMIT University.


Document type: Thesis
Collection: Theses

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Title Mechanisms of g-protein coupled receptor-dependent opening of transient receptor potential vanilloid-4
Author(s) Darby, W
Year 2019
Abstract Transient receptor potential vanilloid-4 (TRPV4) is a non-selective cation channel, involved in thermosensation, mechanosensation and osmosensation. Emerging evidence has identified that the TRPV4 channel is opened by signalling from G-protein coupled receptors (GPCR). TRPV4 is widely expressed; we focused on responses identified in pain sensing neurons, kidney tubule epithelium and the vascular endothelium. The channel has been linked to several pathophysiologies, such as mechanical hyperalgesia and peripheral oedema. TRPV4 integrates a wide range of stimuli which suggests that direct pharmacological modulation of TRPV4 may be difficult.

We aimed to determine the signalling from GPCRs that open TRPV4 channels, as these signalling pathways have not been identified and may prove better targets for pharmacological intervention in TRPV4 related pathophysiologies than modulation of TRPV4.

To assess these the ability of GPCRs to open TRPV4 channels in human embryonic kidney cells (HEK) were used as they endogenously expressed the pro-inflammatory GPCR protease activated receptor-2 (PAR2) and can be stably transfected with hTRPV4 (HEK+TRPV4) using the T-REx inducible system. It was shown that stimulation of PAR2 with its peptide agonist PAR2-activating peptide (PAR2-AP) released intracellular calcium from stores in HEK cells and opened the TRPV4 channel in HEK+TRPV4 cells. It was demonstrated that blockade of the G-alpha q pathway inhibited the release of intracellular calcium from stores in both HEK and HEK+TRPV4 but did not inhibit the PAR2-dependent opening of TRPV4 in HEK+TRPV4 cells. This indicated that PAR2-dependent opening of TRPV4 does not require release of calcium from intracellular or G-alpha q signalling.

Therefore to discover the signalling pathways involved in the PAR2-dependent opening of TRPV4 we developed and optimised a screening process using short interfering ribonucleic acids (siRNAs) to block protein expression  firstly of protein kinases, as the PAR2-dependent opening of TRVP4 has evidence that it is kinase dependent and secondly, of G-proteins and their associated signalling molecules.

The screen of the kinome (the expression of the current known kinases in the human genome) identified with high confidence four siRNAs, siBUB1, siITPK1, siMAPK13 and siITPK1 that were able to interfere with the PAR2-dependent opening of TRPV4 channels, indicating that these proteins were crucial for this signalling process. These results were supported by the qPCR analysis of messenger RNA (mRNA) expression, identifying that all four proteins were expressed in HEK cells, but only treatment with siBUB1, siMAPK13 and siWNK4 caused a significant reduction in the expression of mRNA. Therefore, it was concluded that expressions of the kinases BUB1, MAPK13 and WNK4 are required for the PAR2-dependent opening of TRPV4.

The screen of G-proteins and associated signalling molecules identified that treatment of HEK+TRPV4 cells with siGNA13, siGNG8, siPLA2G4A and siPLCXD3 were effective at blocking the PAR2-dependent opening of TRPV4. It was determined by qPCR detection of mRNA expression that GNA13, GNG8 and PLA2G4A but not PLCXD3 were expressed in the tested HEK cells and that treatment with siRNA for GNA13, GNG8 and PLA2G4A caused a significant reduction of mRNA expression, which confirmed that expression of these proteins are required for the PAR2-dependent opening of TRPV4.

The previous results identified molecules which were previously unknown to participate in the signalling process between PAR2 and TRPV4. To determine the physiological role of the receptor-dependent opening of TRPV4 channels we investigated the ability of other GPCRs, with evidence of involvement in the opening of TRPV4, to open TRPV4 in this HEK cell model. Therefore we tested pro-inflammatory receptors neurokinin 1 (NK1), bradykinin 1, 2 (BK1, BK2) and prostaglandin receptor EP4 (PGTR4). It was identified that stimulation of NK1 with substance P opened TRPV4, as did stimulation of BK2 with bradykinin. These results indicated that these receptors can open TRPV4 channels in HEK cells and expands the potential to assess receptor-dependent opening of TRPV4 channels in dorsal root ganglion neurons.

The ability of adenosine triphosphate (ATP)-dependent GPCRs, called purinergic receptors (P2Y), to open TRPV4 channels were also assessed in HEK293 cells. It was determined that P2Y2 and P2Y11, but not P2Y12, were able to transduce signals required to open TRPV4 when stimulated with their ligand, ATP. The differential ability of these similar receptors; P2Y2, P2Y11 and P2Y12 to open TRPV4 channels demonstrated that stimulation of these receptors in the same HEK cells caused  calcium release from intracellular stores, but the signalling pathway required for GPCR-dependent opening TRPV4 is separate. This evidence confirms earlier experiments in this thesis identifying that GPCR-dependent opening of TRPV4 does not require G-alpha q signalling but has confirmed this with a different GPCR to PAR2.

Activation of the muscarinic receptor M3 (M3R) with its ligand acetyl-choline (ACh) stimulated TRPV4 channel opening in the HEK293 cell model. This finding implicated the vascular endothelium as a potential setting for the receptor-dependent opening of TRPV4.
The final investigation of this thesis assessed the ability of GPCRs in the vascular endothelium of rat cremaster arterioles to open TRPV4 channels. It was shown that stimulation of TRPV4 with its specific agonist GSK1016790A caused an endothelium-dependent vasodilatation that was abolished by the TRPV4 specific antagonists HC067047 and GSK2193874, and inhibition of small and intermediate calcium-dependent potassium channels (SKCa and IKCa). These results demonstrated that TRPV4-dependent vasodilatation is dependent on endothelial derived hyperpolarising factor (EDH).

We then determined that TRPV4 function was altered by shear stress on the endothelium. TRPV4-dependent vasodilatation was sensitised to GSK1016790A after the endothelium had been exposed to shear stress and it was identified that ACh-induced vasodilatation was only sensitive to TRPV4 inhibition after exposure to shear stress. This indicates that the function of TRPV4 was significantly altered in the endothelium with shear stress and that the receptor-dependent opening of TRPV4 required the integration of shear stress into the system.

In conclusion, the results presented in this thesis have identified previously unknown signalling molecules in the receptor-dependent opening of TRPV4. It has also identified the complex nature of TRPV4 signalling in the physiological setting of the vascular endothelium, as TRPV4 integrates many different stimuli to regulate its function. Therefore, the molecules crucial to the receptor-dependent opening of TRPV4 identified could prove to be useful targets for future therapeutic intervention of TRPV4 related pathophysiologies.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Health and Biomedical Sciences
Subjects Basic Pharmacology
Receptors and Membrane Biology
Keyword(s) TRPV4
ion channels
GPCR
intracellular signalling
receptor-operated channel
vasodilatation
shear stress
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