A DOMAIN CONTRIBUTING TO THE ION-CHANNEL OF ATP-GATED P2X(2) RECEPTORS IDENTIFIED BY THE SUBSTITUTED CYSTEINE ACCESSIBILITY METHOD

P2X receptors are a family of ATP-gated ion channels thought to have i ntracellular N and C termini and two transmembrane segments separating a large extracellular domain. We examined the involvement of the seco nd putative transmembrane domain (TM2) of the P2X(2) subunit in ion co nduction, using the substituted cysteine accessibility method (SCAM), This method tests the ability of hydrophilic reagents such as Ag+ or t he methanethiosulfonates to modify covalently the sulfhydryl side chai ns exposed to aqueous environments, ATP-gated current was measured in HEK293 cells transiently expressing either wild-type or functional mut ant P2X(2) receptors containing a cysteine substitution in or around T M2. Application of Ag+ to gating channels had no sustained effect on w ild-type P2X(2) (WT) but irreversibly altered whole-cell currents in 1 5 mutants, By contrast, bath application of (2-aminoethyl)methanethios ulfonate (MTSEA) to closed channels inhibited 8 of the 15 residues aff ected by Ag+ when the channel was gating. Inhibition of the closed cha nnel was prevented in seven of eight mutants when membrane-permeant MT SEA was scavenged by 20 mM intracellular cysteine, indicating that the se seven mutants lie on the intracellular side of the channel gate. Fu rther, MTSEA inhibited current through G342C in the absence of intrace llular cysteine but augmented the current when cysteine was present, s uggesting that this residue may be part of the gate, Taken together, t he data help to the identify a functional domain of the channel pore b y mapping residues on either side of the channel gate.