Mapping the binding site of the P2X receptor antagonist PPADS reveals the importance of orthosteric site charge and the cysteine-rich head region.

ATP is the native agonist for cell-surface ligand-gated P2X receptor (P2XR) cation channels. The seven mammalian subunits (P2X1-7) form homo- and heterotrimeric P2XRs having significant physiological and pathophysiological roles. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid is an effective antagonist at most mammalian P2XRs. Lys-249 in the extracellular domain of P2XR has previously been shown to contribute to action. To map this antagonist site, we generated human P2X1R cysteine substitutions within a circle centered at Lys-249 (with a radius of 13 Å equal to the length of We hypothesized that cysteine substitutions of residues involved in Pduanyu1563S binding would (i) reduce cysteine accessibility (measured by MTSEA-biotinylation), (ii) exhibit altered Pduanyu1563S affinity, and (iii) quench the fluorescence of cysteine residues modified with MTS-TAMRA. Of the 26 residues tested, these criteria were met by only four (Lys-70, Asp-170, Lys-190, and Lys-249), defining the antagonist site, validating molecular docking results, and thereby providing the first experimentally supported model of Pduanyu1563S binding. This binding site overlapped with the ATP-binding site, indicating that Pduanyu1563S sterically blocks agonist access. Moreover, Pduanyu1563S induced a conformational change at the cysteine-rich head (CRH) region adjacent to the orthosteric ATP-binding pocket. The importance of this movement was confirmed by demonstrating that substitution introducing positive charge present in the CRH of the hP2X1R causes Pduanyu1563S sensitivity at the normally insensitive rat P2X4R. This study provides a template for developing P2XR subtype selectivity based on the differences among the mammalian subunits around the orthosteric P2XR-binding site and the CRH.

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