- Title
- Mechanosensitivity of the TRPC6 ion channel
- Creator
- Nikolaev, Yury A.
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2018
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- 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 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 or its activation is mediated via other mechanosensitive membrane structures, such as the cytoskeleton and/or the extracellular matrix. The aim of this study is to determine whether TRPC6 is an inherently mechanosensitive (MS) ion channel. 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. Second, using a stretch device, 15% isotropic stretch was applied to the intact cells attached to the bottom of stretchable PDMS chambers and calcium entry via TRPC6 ion channels was demonstrated. The effect, suggested mechanosensitive nature of TRPC6 channel in the 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.
- Subject
- TRP ion channels; mechanosensitive ion channels; TRPC6; cardiac hypertrophy; cardiomyocyte
- Identifier
- http://hdl.handle.net/1959.13/1387381
- Identifier
- uon:32596
- Rights
- Copyright 2018 Yury A. Nikolaev
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 11 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 246 KB | Adobe Acrobat PDF | View Details Download |