Figure 2.

Na⁺/H⁺ exchanger activity in parental DU 145 cells, in NHE1-knockout cells (NHE1-KO), and in knockout cells that had NHE1 reintroduced (NHE1-RI). (A–D) Examples of traces of activity; these illustrate recovery from an acutely induced acid load. The intracellular pH was recorded in cells transiently acidified using ammonium chloride addition. Periods when NH₄Cl, NaCl, and Na-free solution were added are indicated. (A) wild-type DU 145cells; (B) KO cells; (C) wild-type cells recovered from acid load in the absence of Na⁺; (D) RI: KO cells with NHE1 reintroduced (knock-in). (E) Bar graph showing a summary of the initial rate of recovery; this was measured relative to the wild-type protein and recorded as delta pH/s. n ≥ 6, mean +/− SE. A pH-sensitive indicator dye, BCECF, was used to measure changes in pH_i after the acute acid load induced by the addition and withdrawal of ammonium chloride. The activity of NHE1 was calculated from the changes over the first 20 s of recovery from the acid load, and was expressed as ΔpH/s. Where indicated in (E), 10 µM EMD87580 (EMD) was included in the recoveries to confirm that activity was from Na⁺/H⁺ exchanger activity.