The role of cytoskeletal F-actin in homomeric and heteromeric ∆N-TRPV1 and TRPV4 mechanotransduction.

Author: Stewart, Alex

Date: 2021

Publisher: University of Otago

Type: Thesis

Link to this item using this URL: http://hdl.handle.net/10523/12477

Abstract

Hypertension is one of the leading health concerns facing our population today, however the mechanisms underlying it are not fully understood. This highlights the need to investigate other potential regulators of blood pressure. One regulator of blood pressure is the magnocellular neurosecretory cells (MNC) in the hypothalamus which control the release of vasopressin (AVP) to increase blood pressure. MNCs are intrinsically osmosensitive, so can be activated by changes in osmolality which cause cells to shrink (hyperosmolality) or to swell (hypoosmolality). The intrinsic osmosensitivity of MNCs are controlled by transient receptor potential vanilloid (TRPV) channels. The present study focused on TRPV4, which is active during hypoosmolality, and ∆N-TRPV1, which is active during hyperosmolality. Although TRPV4 and ∆N-TRPV1 have both shown the potential to form heteromers, there is limited research regarding this and whether this could contribute to the mechanical activation of TRPV channels. Therefore, the aim of the present study was to determine the activation of homomeric and heteromeric ∆N-TRPV1, TRPV4 during mechanical stress and to identify whether changes in the F-actin cytoskeleton affect their mechanical activation. Experiments were conducted on Human Embryonic Kidney cells (HEK293) transfected with ∆N-TRPV1, TRPV4 or both ∆N-TRPV1 and TRPV4 and incubated for 10-20 hours. The activity of ∆N-TRPV1, TRPV4 and putative ∆N-TRPV1/TRPV4 was evaluated using single-channel patch-clamp in a cell attached configuration. The channels were investigated with and without pressure using the parameters of current-amplitude, conductance, open probability (NPo) and area under the curve (AUC). Pressure was applied through the recording pipette in the form of either positive (30 cmH2O) or negative (-30 cmH2O) pressure via a water column. In order to investigate the effect of the F-actin cytoskeleton on the channels Cytochalasin D (CytD) was applied to the cells for 40 minutes prior to recordings. Channels were then subjected to single-channel patch clamp and assessed using the same parameters as the untreated channels. Evidence regarding the potential of ∆N-TRPV1 and TRPV4 subunits to combine to form a heteromer was indicated by the localisation of ∆N-TRPV1 and TRPV4 DNA in HEK293 cells. This evidence was further supported by the different current-amplitudes and conductance at 0 cmH2O. This indicated likely evidence of ∆N-TRPV1 and TRPV4 subunits being able to combine to form a functional channel in a HEK293 cell. ∆N-TRPV1 and TRPV4 were both shown to be mechanically activated by cell shrinkage, whereas putative ∆N-TRPV1/TRPV4 showed variable changes to mechanical activity during cell shrinkage. Neither ∆N-TRPV1, TRPV4 or ∆N-TRPV1/TRPV4 had any significant evidence to indicate mechanoactivation to cell swelling. F-actin depolymerisation was shown to reduce the current-amplitude and conductance of both homomeric and heteromeric ∆N-TRPV1 and TRPV4. Putative ∆N-TRPV1/TRPV4 was shown to have an increase in mechanosensitivity to positive pressure but the mechanosensitivity of ∆N-TRPV1 and TRPV4 was decreased when F-actin was depolymerised. While these findings suggest that ∆N-TRPV1 and TRPV4 can form a mechanically sensitive channel in HEK293 cells, whether this channel is present in MNCs remains unknown. Furthermore, as the polymerisation of F-actin was shown to impact the mechanosensitivity of ∆N-TRPV1/TRPV4, putative ∆N-TRPV1/TRPV4 has the potential to play a role in AVP release during hypertension.

Subjects: New Zealand, TRPV4, ∆N-TRPV1

Citation: ["Stewart, A. (2021). The role of cytoskeletal F-actin in homomeric and heteromeric ∆N-TRPV1 and TRPV4 mechanotransduction. (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/12477"]

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