Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599054
Title: Understanding the regulation of ion flow through P2X receptor ion channels
Author: Fisher, J. A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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Abstract:
The cytosolic C-terminal tails of P2X2 subunits were labelled with either cyan or yellow fluorescent proteins (CFP and YFP, respectively). Co-expression of these subunits in human embryonic kidney cells resulted in functional P2X receptors at the plasma membrane that underwent FRET with an efficiency of 34 %. Time-resolved measures of FRET suggested that conformational changes in the C-terminal tails of P2X2-C/P2X2Y receptors were correlated with agonist and time dependent increases in permeability. Similar FRET based measures, suggestive of conformational changes, were not observed in the C-terminal tails of P2X receptors that did not show agonist and time dependent increases in permeability. Furthermore, tethering the P2X2 C-terminal tail prevented permeability changes and abolished conformational changes in the C-terminal tails. Cleavage of the tether restored both phenotypes. Previously generated and characterised α4 and β2 and P2X2 subunits were co-expressed in mammalian cells and shown to undergo cross-inhibition. FRET was used to show that, within ~ 100 nm of the plasma membrane, fluorophore labelled P2X2 and α4β2 nACh receptors were positioned within ~ 80 Å of each other. The FRET signal between these two receptors was greater when the β2 subunit of α4β2 nACh receptor was fluorophore labelled, rather than the α4 subunit, suggesting a preferential orientation for the interaction. This interaction was also measured in hippocampal neurons. In summary the data indicate that the C-terminal tails of P2X2 receptors undergo conformational changes associated with a specific functional property. In addition the C-terminal tails of P2X2 receptors undergo significant spatial interactions with β2 subunits in α4β2 nicotinic acetylcholine receptors. Overall, FRET appears to be a useful complementary approach to study P2X receptors.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.599054  DOI: Not available
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