A Single Amino Acid Mutation Turns a P2X3 Antagonist into an Agonist

Abstract

Purinergic P2X3 receptors (P2X3Rs) are ATP-gated cation channels expressed mainly in sensory neurons. Suramin inhibits P2X3R currents of several species and provides a useful tool for in vitro studies of P2X antagonism. A rhesus homolog of P2X3R was cloned, stably expressed, and pharmacologically characterized. Suramin was a more potent inhibitor of rhesus P2X3R (IC50: 0.4 μM) than of human P2X3R (IC50: 8.6 μM). Surprisingly, suramin activated rhesus P2X3R at higher concentrations (EC50: 4.7 μM). In contrast, suramin did not activate human P2X3R at similar concentrations. Other than differences in suramin agonism, the kinetic and pharmacological profile of rhesus and human P2X3Rs were similar. To investigate the molecular basis of suramin agonism with P2X3R, we generated single-point mutations in the P2X3R. Only four amino acid differences exist between rhesus and human P2X3Rs (S67F, L127F, L144F, and T162M). Mutant receptors were transiently expressed in HEK293 cells and their currents measured using an automated patch clamp instrument (PatchXpress, MDS Analytical Technologies, USA). A single amino acid mutation of human P2X3R (S67F) allowed suramin to act as an agonist and increased the potency of suramin. A corresponding mutation (F67S) in the rhesus P2X3R resulted in a loss of suramin agonism and a decrease of its inhibitory activity. This suggests that position 67 is critical for suramin modulation of P2X3R activity and that inhibition and activation by suramin may be mechanistically related.