Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598546
Title: Functional analysis of IP3 receptors
Author: Ding, Z.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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Abstract:
I developed a high-throughput fluorescence polarisation (FP) binding assay using fluorescein-IP3 and purified N-terminal fragments of IP3R to examine the thermodynamics of ligand binding to IP3R. I demonstrate that at 4°C, equilibrium competition binding using 3H-IP3 and the FP assay provided similar estimates of the equilibrium dissociation constants (KD) for a variety of ligands. I showed that the IBC alone is sufficient for high-affinity binding of adenophostin A (AdA). Similar amounts of binding energy are diverted into rearranging the SD for IP3 and AdA, but they are distinguished by their binding enthalpy and entropy changes. I revealed that the enthalpy and entropy changes of the binding of 2-O-modified IP3 analogues are different, despite their similar free energy changes. These results prompted me to propose a new model to explain partial agonism of IP3R. Different cell-surface receptors have been reported to evoke Ca2+ release from different IP3-sensitive Ca2+ stores. I examined this phenomenon in fura 2-loaded HEK cells. Combined maximal concentrations of ATP and carbachol (CCh) evoked a Ca2+ response that was larger than the response to either alone, but smaller than the sum of the responses. In the absence of extracellular Ca2+, ATP evoked a Ca2+ release that was significantly larger than CCh in cells pretreated with CCh, but smaller than the ATP response in naïve cells. In the absence of extracellular Ca2+, CCh evoked a Ca2+ release that was significantly larger than ATP in cells pretreated with ATP, but smaller than the CCh response in naïve cells. These results suggest the existence of discrete agonist-specific Ca2+-stores that partially overlap.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.598546  DOI: Not available
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