Constantine, Maryrose (2016) Structural and Functional Characterisation of Heterologously Expressed Human Transient Receptor Potential Cation Channel, Subfamily M, Member 4 (TRPM4). PhD thesis, Victor Chang Cardiac Research Institute & Faculty of Medicine, UNSW Australia.
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Maryrose Constantine _PhD Thesis UNSW 2016.pdf Download (4MB) | Preview |
Abstract
Mutation, irregular expression and sustained activation of TRPM4 have been linked to various cardiovascular diseases, including ischemia-reperfusion injury, stroke, arrhythmia (6% Brugada cases), cardiac conduction disorders and hypertension. However, to date much remains unknown about its structure. Here a heterologously expressed human TRPM4-GFP fusion protein was purified, then its supramolecular structure and functionality was examined. The oligomeric state of TRPM4-GFP in detergent micelles was characterised using number and brightness analysis, crosslinking studies, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data demonstrate that TRPM4 is tetrameric, which is the case for other TRP channels studied to date. To assess the functionality of the protein we have obtained single-channel recordings from hTRPM4-eGFP proteoliposomes, which demonstrate inhibition by Flufenamic acid (FFA), suggesting that the channels are fully functional upon reconstitution. The fact that purified TRPM4-GFP maintains a tetrameric form in detergent and exhibits characteristic channel activity indicates that the channel protein we have produced is functional. This body of work will enable future more detailed examination of the structural and functional properties of this therapeutically significant channel.
| Item Type: | Thesis (PhD ) |
|---|---|
| Additional Information: | This work can be used in accordance with the Creative Commons BY-NC-ND license (http://creativecommons.org/licenses/by-nc-d/4.0/). Supervisors: Prof Boris Martinac, Faculty of Medicine, UNSW & VCCRI; Dr Charles Cranfield, School of Life Sciences, University of Technology Sydney.; Prof Chu Kong Liew, Victor Chang Cardiac Research Institute, Sydney. |
| Subjects: | R Medicine > R Medicine (General) |
| Depositing User: | Repository Administrator |
| Date Deposited: | 29 Apr 2016 03:59 |
| Last Modified: | 29 Apr 2016 04:00 |
| URI: | https://eprints.victorchang.edu.au/id/eprint/428 |
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