Christian Sattler, Ralf Schmauder, Tina Schwabe, Andrea Schweinitz, Christopher Unzeitig, Frank Schwede, Maik Otte, Klaus Benndorf
Journal of neurochemistry 2020 AugIonotropic purinergic receptors (P2X receptors) are non-specific cation channels that are activated by the binding of ATP at their extracellular side. P2X receptors contribute to multiple functions, including the generation of pain, inflammation, or synaptic transmission. The channels are trimers and structural information on several of their isoforms is available. In contrast, the cooperation of the subunits in the activation process is poorly understood. We synthesized a novel fluorescent ATP derivative, 2-[DY-547P1]-AET-ATP (fATP) to unravel the complex activation process in P2X2 and mutated P2X2 H319K channels with enhanced apparent affinity by characterizing the relation between ligand binding and activation gating. fATP is a full agonist with respect to ATP that reports the degree of binding by bright fluorescence. For quantifying the binding, a fast automated algorithm was employed on human embryonic kidney cell culture images. The concentrations of half maximum occupancy and activation as well as the respective Hill coefficients were determined. All Hill coefficients exceeded unity, even at an occupancy <10%, suggesting cooperativity of the binding even for the first and second binding step. fATP shows promise for continuative functional studies on other purinergic receptors and, beyond, any other ATP-binding proteins. © 2019 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.
Christian Sattler, Ralf Schmauder, Tina Schwabe, Andrea Schweinitz, Christopher Unzeitig, Frank Schwede, Maik Otte, Klaus Benndorf. Relating ligand binding to activation gating in P2X2 receptors using a novel fluorescent ATP derivative. Journal of neurochemistry. 2020 Aug;154(3):251-262
PMID: 31883343
View Full Text