Functional analysis of IP3 receptors

• Main Author: Ding, Z.
• Published: University of Cambridge 2010
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598546

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 (K_D) 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.