Contribution of the region Glu181 to Val200 of the extracellular loop of the human P2X1 receptor to agonist binding and gating revealed using cysteine scanning mutagenesis(1)

At the majority of mutants in the region Glu181-Val200 incorporating a conserved AsnPheThrΦΦxLys motif cysteine substitution had no effect on sensitivity to ATP, partial agonists, or methanethiosulfonate (MTS) compounds. For the F185C mutant the efficacy of partial agonists was reduced by ∼ 90% but there was no effect on ATP potency or the actions of MTS reagents. At T186C, F188C and K190C mutants ATP potency and partial agonists responses were reduced. The ATP sensitivity of the K190C mutant was rescued towards WT levels by positively charged (2-aminoethyl)methanethiosulfonate hydrobromide and reduced by negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate. Both MTS reagents decreased ATP potency at the T186C mutant, and abolished responses at the F195C mutant. (32)P-2-azido ATP binding to the mutants T186C and K190C was sensitive to MTS reagents consistent with an effect on binding, however binding at F195C was unaffected indicating an effect on gating. The accessibility of the introduced cysteines was probed with (2-aminoethyl)methanethiosulfonate hydrobromide-biotin, this showed that the region Thr186-Ser192 is likely to form a beta sheet and that accessibility is blocked by ATP. Taken together these results suggest that Thr186, Phe188 and Lys190 are involved in ATP binding to the receptor and Phe185 and Phe195 contribute to agonist evoked conformational changes.