Characterization of P2X4-pHluorin constructs in HEK-293 cells. (A) The bar graph summarizes the average data for peak 100-µM ATP-evoked currents at −60 mV for HEK-293 cells expressing each of the indicated constructs. All of the pHluorin constructs were compared with WT P2X4 using a Dunnett one-way ANOVA test. With the exception of P2X4-pHluorin123, they all displayed reduced currents. (B) Representative confocal images of HEK-293 cells expressing the three indicated constructs. Note that no membrane localization was observed for P2X4-pHluorin207. (C) Bar graph summarizes the fluorescence intensity of HEK-293 cells expressing the indicated constructs; all of the data were acquired using identical conditions for transfection and imaging. (D) The top panels show representative Western blots for the indicated constructs, whereas the bottom bar graph summarizes the data for P2X4 band intensities relative to β actin. Note that we observed additional bands in the case of P2X4-pHluorin123 and P2X4-pHluorin156. These likely correspond to partial degradation of the protein. Table 1 summarizes statistical data from experiments such as those shown in Fig. 3. Error bars represent mean ± SEM.