Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486083
Title: Functional studies of mammalian ryanodine receptor channels isolated from cardiac muscle and recombinant channels expressed in HEK293 cells
Author: Song, Lele
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2007
Availability of Full Text:
Access from EThOS:
Abstract:
To investigate the relationship between the structure of adenine nucleotides and their effects on the sheep cardiac ryanodine receptor (RyR2), I have compared the effects of diadenosine pentaphosphate (Ap5A), oxidized Ap5A (oAp5A), ATP and adenosine on the function of RyR2 channels reconstituted into artificial membranes. My results suggest a novel high-affinity adenine nucleotide binding site. Ap5A was found to have higher affinity than ATP for the high-affinity activation site. Ap5A and oAp5A were also found to have higher affinity than ATP for the low-affinity activation site. Unlike ATP, the effects ofAp5A and oAp5A on the low-affinity binding site were not fully reversible. A model is suggested to account for the actions of Ap5A and oAp5A binding to the adenine nucleotide binding site on RyR2. I have also characterised the single-channel function of the rabbit recombinant RyR2 expressed in HEK293 cells. The properties of recombinant RyR2 channels are very similar to those of RyR2 channels isolated from sheep cardiac muscle, however, regulation by ATP appears to be altered. This may be due to the absence of accessory proteins, impairment ofATP binding site or a difference between RyR2 species. With a view to investigating the functional role of the carboxyl terminal tail (CTT) region of the rabbit RyR2, this domain was successfully expressed and purified through pSUMO fusion protein expression system. The structure of the fusion protein, however, was not optimal for SUMO protease digestion and I was not able to purify this region of RyR2 without the fusion protein attached. Future experiments therefore require optimization ofthe fusion protein structure or expression ofCTT in a new system.
Supervisor: Not available Sponsor: Not available
Qualification Name: University of Bristol, 2007 Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.486083  DOI: Not available
Share: