On the contribution of the first transmembrane domain to whole-cell current through an ATP-gated ionotropic P2X receptor.

Scanning cysteine mutagenesis was used to identify potential pore-forming residues in and around the first transmembrane domains of ionotropic P2X(2) receptor subunits. Twenty-eight unique cysteine-substituted mutants (R28C-Y55C) were individually expressed in HEK293 cells by lipofection. Twenty-three of these were functional as assayed by application of ATP to transfected voltage-clamped cells. Individual mutants varied in their sensitivity to ATP; otherwise, currents through functional mutant receptors resembled those of the homomeric wild-type (WT) receptor. In five (H33C, R34C, I50C, K53C, and S54C) of 23 functional mutants, coapplication of 30 microm ATP and 500 nm Ag(+) irreversibly inhibited inward current evoked by subsequent applications of ATP alone. These inhibitions did not result in a lateral shift in the agonist concentration-response curve and are unlikely to involve a modification of the agonist binding site. Two (K53C and S54C) of the five residues modified by Ag(+) applied in the presence of ATP when the channels were gating were also modified by 1 mm (2-aminoethyl)methanethiosulfonate applied in the absence of ATP when the channels were closed. These data suggest that domains near either end of the first transmembrane domain influence ion conduction through the pore of the P2X(2) receptor.