Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X(2) receptor

The first hydrophobic segment of the rat P2X(2) receptor extends from resid ue Leu(29) to Val(51), In the rat P2X(2) receptor, we mutated amino acids i n this segment and adjoining flanking regions (Asp(15) through Thr(60)) ind ividually to cysteine and expressed the constructs in human embryonic kidne y cells. Whole-cell recordings were used to measure membrane currents evoke d by brief (2-s) applications of ATP (0.3-100 muM). Currents were normal ex cept for Y16C, R34C, Y43C, Y55C, and Q56C (no currents but normal membrane expression by immunohistochemistry), Q37C (small currents), and F44C (norma l current but increased sensitivity to ATP, as well as cup-methylene-ATP), We used methanethiosulfonates of positive, negative, or no charge to test t he accessibility of the substituted cysteines. D15C, P19C, V23C, V24C, G30C , Q37C, F44C, and V48C were strongly inhibited by neutral, membrane-permean t methanethiosulfonates. Only V48C was also inhibited by positively and neg atively charged methanethiosulfonates, consistent with an extracellular pos ition; however, accessibility of V48C was increased by channel opening. V48 C could disulfide with I328C, as shown by the large increase in ATP-evoked current caused by reducing agents. The results suggest that Val(48) at the outer end of the first hydrophobic segment takes part in the gating movemen t of channel opening.