Figure 6.
Gprc5c increases NHE3 exchanger activity at alkaline extracellular pH. Acid-loaded HEK293T cells were transfected with either empty vector [control (ctl)] or Gprc5c (postsignal peptide) and subsequently loaded with BCECF. The initial rate of sodium-dependent alkalization with which the cells recover from acidification (slope) is a measure of sodium-dependent proton exchanger activity. A) Representative pHi tracing of CTL and Gprc5c-transfected cells at pH 7.4. Bars on the graph indicate when the appropriate solutions were applied to the cells. K+ clamp buffer containing 10 μm nigericin was adjusted to pH 6.0, 6.8, or 7.4 and applied to each coverslip at the end of recording to calibrate the pHi. When the pH of the extracellular solution was pH 7.4, we did not observe any difference in exchanger activity between ctl and Gprc5c-transfected cells. B) When the pH of extracellular solution is 8.0, we find that the sodium proton exchanger activity is higher in Gprc5c-transfected (vs. empty vector-transfected) cells. C) Addition of 100 nM tenapanor (NHE3-specific inhibitor) abolished the increase in sodium proton activity seen in Gprc5c-transfected cells at pH 8.0. D–F) Summary of the sodium proton exchanger activity of cells calculated from the slope and normalized to the averaged activity of empty vector-transfected cells at pH 7.4 (D), pH 8 (E), and pH 8 with 100 nM tenapanor (F). The sodium proton exchanger activity of Gprc5c-transfected cells at pH 8.0 is significantly increased compared with empty vector-transfected cells. G) An estimation of the tenapanor-sensitive NHE3 activity at pH 8 between empty vector and Gprc5c-transfected cells. For this graph, the tenapanor-sensitive activity at pH 8 for both empty vector and Gprc5c is plotted; all values were normalized to empty vector pH 8 (without tenapanor). Each measurement was performed on separate coverslips with independent transfections (CTL pH 7.4: n = 4; Gprc5c pH 7.4: n = 4; ctl pH 8.0: n = 7; Gprc5c pH 8.0: n = 7; ctl pH 8.0 + tenapanor: n = 3; Gprc5c pH 8.0 + tenapanor: n = 3). *P < 0.05 (Student’s t test).