FIGURE 2.

¹⁰³⁸AA¹⁰³⁹ and ¹⁰⁴⁸AA¹⁰⁴⁹ proteins are not expressed at the cell surface. Immunolocalization studies of wild-type and mutated NKCC2 proteins. Cells were transiently transfected with Myc-tagged wild-type and mutant NKCC2 proteins, as indicated. Forty-eight hours post-transfection, membrane proteins of confluent OKP cells (A) or HEK cells (B) were biotinylated at 4 °C with the biotinylation reagent sulfo-NHS-SS-biotin. Then the monolayers were fixed and stained for Myc tag NKCC2 protein (Texas red) and cell surface biotin (avidin-Cy2). The stained specimens were evaluated by confocal microscopy. Optical sections (xy) at the cell surface are depicted for Texas red (red) or Cy2 channel (green), and a merged channel. The yellow color (merged image) represents co-localization of NKCC2 with biotinylated membranes, and therefore indicates correct targeting to the cell surface. The white bars represent 5 μm. C, measurement of Na-K-2Cl co-transport activity in OKP cells expressing WT NKCC2 or mutant proteins; ¹⁰³⁸LL¹⁰³⁹/AA and ¹⁰⁴⁸LI¹⁰⁴⁹/AA. Mean initial rate of pH_i recovery (dpH_i/dt) from NH₄⁺-induced alkaline load. Each bar represents the mean ± S.E. rates of cell pH recovery (dpH_i/dt, pH units/min) under different experimental conditions (mock cells, cells expressing Myc-NKCC2, cells transfected with Myc-¹⁰³⁸LL¹⁰³⁹/AA and cells transfected with ¹⁰⁴⁸LI¹⁰⁴⁹/AA). *, p < 0.05 versus WT NKCC2 (Mock, n = 7; WT NKCC2, n = 6; ¹⁰³⁸LL¹⁰³⁹/AA, n = 8 and ¹⁰⁴⁸LI¹⁰⁴⁹/AA, n = 7).