Abstract

Lysine residues near each end of the receptor ectodomain (in rat P2X₂ Lys⁶⁹ and Lys³⁰⁸) have been implicated in ATP binding to P2X receptors. We recorded membrane currents from human embryonic kidney cells expressing P2X subunits and found that lysine-to-alanine substitutions at equivalent positions in the P2X₃ receptor (Lys⁶³ and Lys²⁹⁹) also prevented channel function. Heteromeric P2X_2/3 receptors are formed when P2X₂ and P2X₃ subunits are expressed together; they can be distinguished by their relatively sustained response to αβ-methylene-ATP. By coexpression of wild-type P2X₃ and mutated P2X₂ subunit, we found that the heteromeric P2X_2/3 channel functioned normally when either lysine in the P2X₂ subunit was mutated to alanine (i.e., [K69A] or [K308A]) but not when both lysines were mutated to alanine (i.e., [K69A, K308A]). However, coexpression of wild-type P2X₂ with a mutated P2X₃ subunit ([K68A] or [K299A]) produced no functional heteromers. The rescue of the single lysine mutant P2X₂ subunit by wild-type P2X₃ (but not the converse) suggests that the heteromeric channel contains one P2X₂ and two P2X₃ subunits and that the receptor functions essentially normally as long as two subunits are not mutated. The failure to rescue function in the P2X₂ subunit with both lysines mutated by wild-type P2X₃ suggests that these residues from two different subunits interact in agonist binding or channel opening.