Molecular studies of transient receptor potential Vanilloid 3 (TRPV3)
Creators: name: AL-ARYAHI, Sefaa
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310110
subject: Receptors and membrane biology
classificationCode: 310111
subject: Signal transduction
classificationCode: 320906
subject: Peripheral nervous system

description: Transient receptor potential, subfamily V, member 3 (TRPV3) is a calcium (Ca2+) permeable non-selective cation channel. It is mainly expressed in skin keratinocytes. TRPV3 is activated by temperatures ≥ 33 °C and by pungent chemicals such as eugenol, carvacrol and camphor; and by the non-selective synthetic agonist 2-Aminoethoxydiphenyl borate (2-APB). TRPV3 is known to regulate a variety of physiological functions, such as skin barrier formation, hair growth, wound healing, temperature sensing, itch and pain perception. Recent studies have also shown that TRPV3 gain of function mutations are involved in skin diseases. The function of TRPV3 in diseases and skin disorders is not well understood and more research is
required. Therefore, the first aim of this thesis was, to generate and characterise a human TRPV3 expressing cell line. From this study I was able to generate a stable human TRPV3 transfected in human embryonic kidney 293 (HEK293) cells via the tetracycline-inducible system.

Furthermore, it has been reported that TRPV3 is involved in lung cancer and is expressed in human alveolar basal epithelial (A549) cells. The function of TRPV3 in A549 cells has not been investigated. An A549 lung cancer cell line may be useful to characterize TRPV3 channel function and to understand its role in lungs and to complement studies in TRPV3 transfected cells. In addition, our lab has shown that Gprotein coupled receptors (GPCRs) can signal to and open Transient Receptor Potential ion channel, member V4 (TRPV4). Therefore, I investigated whether GPCRs can also
open or sensitise TRPV3 in A549 cells and keratinocytes.

Therefore, the second aim of the study was to characterise keratinocytes and A549 cells using a fluorescent intracellular Ca2+ assay and investigate the potential physiological mechanisms of TRPV3 modulation by determining whether activation of GPCRs can open or sensitise TRPV3 ion channels, as has been shown for some other TRP ion channels. The study demonstrated that GPCRs did not sensitise or open TRPV3 channels. On the other hand, interestingly all the GPCRs investigated desensitised the channel to its non-selective agonist 2-APB.

Selective agonists and antagonists have not been identified for TRPV3, making it difficult to understand and study its physiological activation and modulation. This leads to aim three, which involved identifying and characterising novel agonists. I successfully identified novel agonists and characterised them using a fluorescent intracellular Ca2+ assay.

Repeated heat and chemical stimulation have been shown to potentiate TRPV3 channel activity, unlike other TRP channels which reportedly desensitise with repeat stimulation. However, the mechanisms behind this unique characteristic of TRPV3 gating had not been determined. 
Therefore, in aim four I examined the mechanism by which repeated stimulation augments TRPV3 responses using biotinylation assays to look at the cell surface expression of TRPV3 at different temperatures. The study demonstrated that repeated heat stimulation did not cause an increase in the cell surface expression of TRPV3 therefore, potentiation of TRPV3 by repeat agonist or heat stimulation may be due to phosphorylation of TRPV3 as seen in the mass spectrometry data.

Furthermore, studies have shown that pH 5.6, arachidonic acid and other unsaturated fatty acids can directly potentiate the non-selective agonist 2-APB to its channel TRPV3. TRPV3 is expressed in the endothelium of intact cerebral and cerebellar arteries and therefore is exposed to mechanical force (shear stress) from blood flow. Shear stress is an important physiological stimulus that induces a cellular response in vivo. In vitro, physiological levels of mechanical force can be applied to cultured cells using a microfluidic device. There are no publications investigating the mechanosensitivity of TRPV3. Therefore, I sought to determine if shear stress had any effect on the sensitisation or opening of the TRPV3 ion channel. The
mechanosensitivity study confirmed a novel finding which involves TRPV3 being sensitised by shear stress and that kinase inhibitors further increased this sensitivity of TRPV3 channel to it 2-APB agonist.

In summary, TRPV3 is known to be involved in a number of important physiological processes and dysfunction of this channel is associated with skin diseases, itch and pain. However, due to the lack of pharmacological tools, the physiological mechanisms by which TRPV3 is activated and regulated are not well understood. The purpose of this thesis was to add to the current scientific knowledge of the role of the TRPV3 ion channel in the human body. A novel physiological function of TRPV3 was identified in this thesis which was the role of TRPV3 in mechanotransduction, where shear stress
sensitised the TRPV3 response to it agonist 2APB. Understanding the mechanism of sensitisation will help to explain TRPV3 function in the endothelium, where it is exposed to blood flow and in nerves and skin, where it contributes to itch.