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P2X receptors are a family of ATP-gated ion channels thought to have intracellular N and C termini and two transmembrane segments separating a large extracellular domain. We examined the involvement of the second putative transmembrane domain (TM2) of the P2X₂subunit in ion conduction, using the substituted cysteine accessibility method (SCAM). This method tests the ability of hydrophilic reagents such as Ag⁺ or the methanethiosulfonates to modify covalently the sulfhydryl side chains exposed to aqueous environments. ATP-gated current was measured in HEK293 cells transiently expressing either wild-type or functional mutant P2X₂ receptors containing a cysteine substitution in or around TM2. Application of Ag⁺ to gating channels had no sustained effect on wild-type P2X₂ (WT) but irreversibly altered whole-cell currents in 15 mutants. By contrast, bath application of (2-aminoethyl …