FIGURE 4.

Characterization of site-specific mutants of Leu⁴⁶⁵. A, Western blot of cell extracts of AP-1 cells containing stably transfected Na⁺/H⁺ exchanger mutants or control. Samples were analyzed as described in the legend to Fig. 2A. Numbers below the lanes indicate the values obtained from densitometric scans of both the 110- and 95-kDa bands relative to wild type NHE. Results are typical of three to five measurements. cNHE refers to cysteine-less NHE1. A, D, and K refer to mutant cell lines containing cNHE1 with the Leu⁴⁶⁵ mutations to Ala, Asp, and Lys respectively. B, subcellular localization of TM XI mutants in AP-1 cells. Mutants contained the NHE1 Leu⁴⁶⁵ mutations to Ala, Asp, and Lys as indicated. Sulfo-NHS-SS-biotin-treated cells were lysed and streptavidin-agarose beads were used to bind labeled proteins as described under “Experimental Procedures.” Equal amounts of total cell lysate (T) and unbound intracellular lysate (I) were run on SDS-PAGE and Western blotting with anti-HA antibody identified the NHE1 protein. The percent of the total NHE1 protein found within the plasma membrane is indicated for each mutant. Results are the mean ± S.E. of at least three determinations. C, rate of recovery from an acid load by AP-1 cells stably transfected with cNHE, and TM XI Na⁺/H⁺ exchanger Leu⁴⁶⁵ mutations to Ala, Asp, and Lys. Na⁺/H⁺ exchanger activity was measured after transient induction of an acid load as described in the legend to Fig. 2C. All mutations to cysteine were done in the background of the cysteine-less NHE1 and activities are percent of those of cNHE. Mutations were in the cNHE1 and results are expressed as mean ± S.E. of 8-16 determinations. Results are shown for mean activity of both uncorrected (black) and normalized for surface processing and expression levels (hatched).