Nikolaev, Yury A (2018) MECHANOSENSITIVITY OF THE TRPC6 ION CHANNEL. PhD thesis, Victor Chang Cardiac Research Institute & Faculty of Health, Newcastle University.
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Abstract
Background: The transient receptor potential (TRP) ion channel family is a
diverse group of channels gated by various physical and chemical stimuli. One of the members, TRPC6, is a Ca2+ permeable cation channel, which is expressed in ventricular cardiomyocytes (CMs). TRPC6 is gated via the G-protein-coupled receptor pathway leading to generation of diacylglycerol (DAG), which ultimately activates the ion channel. TRPC6 can also be activated by mechanical force, which for example plays a significant role in mechanotransduction of the heart. However, it is still unclear whether TRPC6 is activated directly by membrane tension (the bilayer mechanism) or its activation is mediated via other mechanosensitive membrane structures, such as the cytoskeleton and/or the extracellular matrix (the tether mechanism).
The aim of the study: To determine whether TRPC6 is an inherently mechanosensitive (MS) ion channel.
Methods and Results: First, mechanosensitivity of TRPC6 was evaluated in
HEK293 cells by stretching the membrane via application of negative pressure
(suction) to a patch pipette. Spontaneously active TRPC6 channel did not respond
to the force. The entire-cell TRPC6 currents were revealed in the whole
cell configuration and the channels were activated by DAG analogue. Second, using a stretch device, 15% isotropic stretch was applied to the intact cells attached
to the bottom of stretchable PDMS chambers and Ca2+ entry via TRPC6
ion channels was demonstrated. The effect, suggested mechanosensitive nature
of TRPC6 channel in HEK293 cells, however, it remained unclear whether
mechanosensitivity is inherent or promoted via other membrane components.
To answer this question, using the purified TRPC6 protein, liposome reconstitution
was carried out. Spontaneous activity of the TRPC6 single channel was
demonstrated in the liposome by the patch clamp. The channel was activated
according to “force-from-lipids” principle; however, application of stretch did
not change the open probability of the channel. Therefore, it has been concluded
that TRPC6 is not stretch activated upon application.
Furthermore, the role of TRPC6 in cardiac hypertrophy was investigated. Immunostaining
of TRPC6 in hypertrophic CMs revealed that the channel migrates
from the intracellular t-tubules to the sarcolemma. Furthermore, the impact
of MS channels on the Ca2+ homeostasis in the CMs was investigated. A
new method was developed, which allowed stretching of hydrogel embedded
CM in multiaxial (isotropic) directions and simultaneous measurement of Ca2+
fluorescence. Both normal and hypertrophic CMs showed a late mechanical response 300s after the stretch. MS channels were only activated after long-term induced stretch, which suggests their mechanoprotective role in the heart.
The main finding of the thesis is that the TRPC6 ion channel is not inherently mechanosensitive since it is unresponsive to membrane stretch; instead it is activated by “force-from-lipids” principle without involvement of any other membrane components. Since abnormal TRPC6 activity is implicated in cardiac hypertrophy, our findings contribute to a better understanding of pathophysiological mechanisms of hypertrophy and may open up new directions for therapeutic strategy.
| Item Type: | Thesis (PhD ) |
|---|---|
| Additional Information: | SUPERVISORS: Prof Boris Martinac, Prof Derek Laver and Prof Dirk van Helden |
| Subjects: | R Medicine > R Medicine (General) |
| Depositing User: | Repository Administrator |
| Date Deposited: | 17 Apr 2019 01:13 |
| Last Modified: | 16 Oct 2020 00:58 |
| URI: | https://eprints.victorchang.edu.au/id/eprint/833 |
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