Mechanosensitivity of TRPV Channels: Implications for Vasopressin Neuron Activity

Vasopressin is synthesised by magnocellular neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and is secreted from the posterior pituitary gland in response to increased plasma osmolality. Vasopressin maintains body fluid balance by promoting renal water retention. Mechanosensitive TRPV channels facilitate vasopressin neuron responses to plasma osmolality and express various TRPV subunits, including ΔN‐TRPV1, TRPV2 and TRPV4. Most TRPV channels express more than one type of TRPV subunit but the subunit composition of the TRPV channels expressed by vasopressin neurons is unknown, as is the potential contribution of any changes in composition to vasopressin neuron function. Here, we tested the hypothesis that a change in TRPV channel composition alters the mechanosensitivity of ΔN‐TRPV1‐containing channels. To test this hypothesis TRPV and vasopressin expression in the SON of female Sprague Dawley rats was visualised using immunohistochemistry and will also be visualised with RNAscope. Xenopus oocytes and HEK293 cells were used for heterologous expression of TRPV channels and membrane current recorded via electrophysiology. TRPV2 was expressed on ~33% of SON vasopressin neurons and TRPV4 was expressed on ~40%. However, the TRPV1 antibody caused non‐specific staining and instead RNAscope will be used. Xenopus oocytes expressing ΔN‐TRPV1, TRPV2 and TRPV4 were recorded via two‐electrode‐voltage‐clamp (TEVC). However, TRPV channels in Xenopus oocytes do not appear to be functional because there was no change in membrane current during application of TRPV antagonists and agonists, independent of injected RNA amount (1–20ng) and incubation time (1–4 days). Transfection of TRPV channels into HEK293 cells with co‐expressed fluorescent protein; ΔN‐TRPV1‐GFP, TRPV2‐mCherry and TRPV4‐CFP was performed. Maximum ΔN‐TRPV1‐GFP expression occurred from 10–20 h, however increased cell death was observed after 24 h. In conclusion, vasopressin neurons express TRPV channels but Xenopus oocytes appear to lack specific cytoskeletal proteins for mechanical gating such as beta‐tubulin. HEK‐293 cells express the required cytoskeletal proteins for TRPV trafficking, membrane tethering and gating and we are in process of testing whether ΔN‐TRPV1‐GFP transfection in HEK‐293 cells forms mechanosensitive channels.