Figure 5.

Analysis of voltage- and [ATP]-dependent gating of tandem trimers with three, two, and one T339S mutant subunit in the pore region of the P2X₂ receptor channel together with WT–WT–WT. An increase in the number of T339S in the trimer had a gradual and additive effect on voltage-dependent gating. (A) Representative macroscopic current recordings from tandem trimers with three, two, and one T339S, evoked by step pulses during the steady state after the application of saturating concentrations of [ATP]. The holding potential was −40 mV. Step pulses from 40 to −160 mV were applied in 20-mV decrements. Tail currents were recorded at −60 mV, and their enlarged images are shown next to the main activation traces. (B) Normalized G-V relationships derived from maximum tail currents at −60 mV fitted with the two-state Boltzmann equation are shown as mean ± SEM at 100 µM [ATP] for WT–WT–WT (red; n = 12), WT–T339S–WT (blue; n = 9), WT–T339S–T339S (magenta; n = 11), and T339S–T339S–T339S (black; n = 6). (C) Ratio of conductance at −60 and −160 mV at 100 µM [ATP] for tandems having three, two, and one T339S mutant subunit and WT–WT–WT is shown by a boxplot using the same color code as in B. Means are shown by a plus sign. The bottom of each box is the 25th percentile, the top is the 75th percentile, and the line in the middle is the median. Whiskers show the minimum and maximum data. *, P < 0.05. (D) Normalized voltage-induced activation traces for tandem trimers with one, two, and three T339S mutation(s) and WT–WT–WT at −160 mV at various concentrations of ATP from the representative traces shown in A (color code is the same as in C). (E) Comparisons of activation time constants at saturating concentrations of ATP for WT–WT–WT (n = 20) and tandem trimers with two (n = 35) and one (n = 25) T339S mutations by fitting the voltage-induced activation phase to a single-exponential function, as explained in Materials and methods. Data are shown by boxplot as in C, using the same color code. *, P < 0.05. As there is no voltage-induced activation for the tandem trimer with three T339S subunits, it was not analyzed. (F) [ATP] dose–response relationships for tandem trimers at −160 mV. EC₅₀ and Hill coefficients are as follows: T339S–T339S–T339S: EC₅₀, 2.4 ± 0.4 µM, and Hill, 1.5 ± 0.3 (n = 7); WT–T339S–T339S: EC₅₀, 6.0 ± 0.4 µM, and Hill, 1.5 ± 0.2 (n = 10); and WT–T339S–WT: EC₅₀, 12.2 ± 1.2 µM, and Hill, 1.5 ± 0.9 (n = 6; color code is the same as in B).