FIGURE 9.
Electrophysiological studies of WT-PCFT, H247A-PCFT, and H281A-PCFT expressed in Xenopus oocytes. pHi changes (upper tracing) and currents (lower tracing) were recorded simultaneously in individual oocytes voltage-clamped at Vh = −90 mV. Oocytes were superfused with ND90 solution (at the indicated pHo; (5.5 blue shading and 4.5 yellow shading), and then MTX was added (gray shaded column). The general protocol was designed to change one solution component at a time: beginning at pHo 7.5 (no shading), change pHo to 5.5 (blue shading), add MTX, remove MTX, change to pHo of 4.5 (yellow shading), add MTX, remove MTX, return to pHo 7.5. A, recordings in an oocyte injected with WT-pcft at pH 5.5 and 4.5; 20 μm MTX was utilized, a concentration that saturates WT-pcft. B, a water-injected control oocyte with the identical protocol on the same day. C, the H281A-PCFT-injected oocytes. The MTX concentration used in this protocol was 100 μm to compensate for the high Kt for this mutant. D and E are the H247A-pcft-injected oocytes. In D, oocytes were exposed to pH 5.5 buffer first then to pH 4.5 buffer. In E, the order of exposure was reversed. In both cases the pH was returned to 7.4 between changes in the acid buffers. Recordings are representative of 3–8 separate experiments per experiment type from at least 3 donor Xenopus oocytes. The y axis in C indicates a range of −100 to −500 nA; the other panels indicate a range of 0 to −300 nA. All five experiments have been scaled so that the “5-min” time bar is identical. The text below the time bar indicates the actual experiment number.