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Relating ligand binding to activation gating in P2X2 receptors using a novel fluorescent ATP derivative
Author(s) -
Sattler Christian,
Schmauder Ralf,
Schwabe Tina,
Schweinitz Andrea,
Unzeitig Christopher,
Schwede Frank,
Otte Maik,
Benndorf Klaus
Publication year - 2020
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.14948
Subject(s) - ionotropic effect , purinergic receptor , receptor , biophysics , ligand (biochemistry) , binding site , ligand gated ion channel , gating , cooperative binding , microbiology and biotechnology , cooperativity , agonist , chemistry , biochemistry , biology , glutamate receptor , ion channel
Ionotropic 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.