Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor.

P2X receptor subunits have intracellular N and C termini, two membrane-spanning domains, and an extracellular loop of about 280 amino acids. We expressed the rat P2X(2) receptor in human embryonic kidney cells, and used alanine-scanning mutagenesis on 30 residues with polar side chains conserved among the seven rat P2X receptor subunits. This identified a region proximal to the first transmembrane domain which contained 2 lysine residues that were critical for the action of ATP (Lys(69) and Lys(71)). We substituted cysteines in this region (Asp(57) to Asp(71)) and found that for S65C and I67C ATP-evoked currents were inhibited by methanethiosulfonates. At I67C, the inhibition by negatively charged ethylsulfonate and pentylsulfonate derivatives could be overcome by increasing the ATP concentration, consistent with a reduced affinity of ATP binding. The inhibitory action of the methanethiosulfonates was prevented by pre-exposure to ATP, suggesting occlusion of the binding site. Finally, introduction of negative charges into the receptor by mutagenesis at this position (I67E and I67D) also gave receptors in which the ATP concentration-response curve was right-shifted. The results suggest that residues close to Ile(67) contribute to the ATP-binding site.