Amino Acid Residues Involved in Gating Identified in the First Membrane-spanning Domain of the Rat P2X2 Receptor

The first hydrophobic segment of the rat P2X2 receptor extends from residue Leu29 to Val51. In the rat P2X2 receptor, we mutated amino acids in this segment and adjoining flanking regions (Asp15 through Thr60) individually to cysteine and expressed the constructs in human embryonic kidney cells. Whole-cell recordings were used to measure membrane currents evoked by brief (2-s) applications of ATP (0.3-100 μM). Currents were normal except for Y16C, R34C, Y43C, Y55C, and Q56C (no currents but normal membrane expression by immunohistochemistry), Q37C (small currents), and F44C (normal current but increased sensitivity to ATP, as well as αβ-methylene-ATP). We used methanethiosulfonates of positive, negative, or no charge to test the accessibility of the substituted cysteines. D15C, P19C, V23C, V24C, G30C, Q37C, F44C, and V48C were strongly inhibited by neutral, membrane-permeant methanethiosulfonates. Only V48C was also inhibited by positively and negatively charged methanethiosulfonates, consistent with an extracellular position; however, accessibility of V48C was increased by channel opening. V48C could disulfide with I328C, as shown by the large increase in ATP-evoked current caused by reducing agents. The results suggest that Val48 at the outer end of the first hydrophobic segment takes part in the gating movement of channel opening.