Fig. 6. Functional characterization of AeNHE3 in a NHE-deficient cell line. (A) Untransfected PS120 cells fail to recover intracellular pH following an acid load, but maintain a near neutral pH in culture medium (DMEM). Cells were challenged with a H⁺ load (+) or left unchallenged (-) and subsequently changed to buffer (135 mmol l^-1 NaCl, HBS, pH 7.4) or culture medium (DMEM) and assayed by ratiometric fluorimetry. (B) Polyclonal cells from two separate transfection experiments (NHE3a and NHE3b) expressing full-length AeNHE3 were subjected to acid load and recovery of intracellular pH was monitored by BCECF fluorescence. (C) A stable clone (clone A2) expressing AeNHE3 was subjected to acid load and changed to buffers containing the indicated concentrations of ions to monitor change in intracellular pH (pH_i) with (+) or without (-) inhibitors of Na⁺/K⁺-ATPase (1 mmol l^-1 ouabain) and Na⁺/K⁺/Cl^- cotransporter (100 µmol l^-1 bumetanide). Experiments with another stable clone (clone B3) produced similar results. (D) The cytoplasmic carboxy terminal of AeNHE3 is not required for its function. A stable clone (NHE C-clone A8) that expresses AeNHE C (lacking carboxy 448 amino acids) was assayed for pH_i recovery after an acid load in the presence or absence of inhibitors as stated in C. Experiments with another stable line (NHE C-clone B7) produced similar results. The buffers also contained 5 mmol l^-1 glucose, 2 mmol l^-1 CaCl₂ and 1 mmol l^-1 MgCl₂. Values are means ± standard error (N=4-8).