Title:
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Functional studies of mammalian ryanodine receptor channels isolated from cardiac muscle and recombinant channels expressed in HEK293 cells
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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.
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