Na+/H+ exchanger activity: Measurement of ion concentration and proton (H+) flux

Intracellular pH (pH_i) was measured via fluorescence using the pH indicator dye, BCECF (2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescerin-acetoxymethyl ester). Briefly, cells were grown to 80% confluence on rectangular glass coverslips in 35 mm dishes in complete media, and either serum-starved or not for 24 hr. post-attachment. BCECF-AM, a cell permeable dye, was added to cells for 20 minutes at 37°C. During this time, cytosolic esterases cleave the AM ester, allowing fluorescent BCECF to become charged, polarized, and cell impermeable; intracellular BCECF fluorescence is thus a measure of pH_i. Cells were then acidified by addition of ammonium chloride (50 mM, 3 min) followed by its rapid withdrawal [54]. For recovery post-acid load, cells were perfused with Na⁺-free and then Na⁺-containing buffers. After measurement of activity, pH_i was calibrated using buffer containing nigericin and high K⁺ at pH 6, 7 and 8. The ratio of BCECF fluorescence with excitation at 440 nm and 502 nm, and emission at 528 nm, was recorded using a PTI Deltascan Illumination System (Photon Technology International, New Jersey, USA). Buffering capacity was calculated and was similar across all cell types (data not shown). NHE1 activity was calculated from the slope of the first 20 sec of recovery of pH_i from acidification (ΔpH_i/sec). Cells were either stimulated (0.2% serum) overnight prior to measuring NHE1 activity, or supplemented with 10% serum (unstimulated). In experiments testing the effect of BI-D1870 on NHE1 activity, cells were additionally treated with 10 μM BI-D1870 overnight. Data were normalized to the wild-type (wtNHE1) control to show relative Na⁺/H⁺ exchange activity between cell types.

Proton affinity was a measure of H⁺ flux (J_H⁺). H⁺ flux was calculated as the product of the rate of change of pH_i over time (ΔpH_i/sec) and the buffering capacity of cells (B mmol • 1⁻¹ • pH unit⁻¹) as previously described [56]. Buffering capacity was determined by varying ammonium chloride concentrations (10 mM, 30 mM, or 50 mM) and measuring intracellular pH and calculating buffering as described earlier [56].

Intracellular and extracellular Na⁺, K⁺, Mg²⁺, and Ca²⁺ concentrations of wtNHE1 and NHE1-mutant cells were determined using inductively coupled plasma optical emission spectroscopy (ICP-OES, iCAP 6000, Thermo Scientific, Canada). Cells were seeded in a 12-well plate, grown to confluence, and either stimulated (0.2% serum) or unstimulated (10% serum) overnight. Cells were thoroughly washed in sodium- and potassium-free buffer and then lysed with 0.5 mL of 0.1% Triton X-100 and 0.2% nitric acid overnight at 4°C with agitation, prior to sonication for 1 min as previously described [57]. Samples and buffer blanks were then diluted 100 times in ultrapure deionized water and filtered to remove any particulate matter. For the ICP-OES analysis, the digestion method used was EPA 3051, with nitric acid at a ratio of 5 mL HNO₃ to 20 mL ultrapure deionized water, using the Xpress Mars Microwave Digestion System (CEM Corp., US).