The Molecular Basis of Antagonism by PPADS at the Human P2X1 Receptor

P2X receptors (P2XRs) activated by ATP are widely expressed throughout the human body and mediate various physiological and pathophysiological roles. Crystal structures have provided a major advance in understanding agonist and subtype selective antagonist actions. However, the molecular basis of antagonism of general antagonists is poorly understood. PPADS is an effective antagonist at most mammalian P2XRs. Previous studies suggested lysine residue 249 (K249) (numbering for P2X1R) was involved in PPADS action. The aim of this study was to determine the PPADS binding site in a molecular model of the human P2X1R based on the zebra fish P2X4R (zfP2X4R) crystal structure. Contributions of individual residues in a ring centred on K249 with a radius of the length of PPADS were investigated by cysteine mutagenesis. The effect of their cysteine substitutions on accessibility following PPADS binding and on PPADS sensitivity were tested. A cluster of positively charged residues (K70, K190 and K249) at the orthosteric pocket showed decreases in both accessibility and sensitivity to PPADS, suggesting they are directly involved in binding of the antagonist. These data allow validation of molecular docking to provide the first model of PPADS binding. Some residues outside the orthosteric area showed decreased accessibility following PPADS binding but on change in antagonist sensitivity, indicating PPADS binding induced significant conformational changes from the apo state. In addition, the charge and conformational changes at the cysteine rich head (CRH) region also contributed to antagonist action by showing a mutation at a positively charged residue (K138C) increased accessibility following PPADS binding and decreased PPADS sensitivity. In summary, this thesis has advanced the understanding of antagonist PPADS action and provided a template to develop subtype selectivity based on the differences between subunits around the orthosteric P2XR binding site and the CRH region.