Table 1.
Oligonucleotides used for site-directed mutagenesis (forward primer).
Fig 1.
Wild type (WT) NHE1 protein expression and characterization.
A, Model of the NHE1 protein within the plasma membrane [18]. The approximate position of the shortened NHE1 proteins is indicated in red. B, Western blot of whole cell lysates of stably transfected cell lines. Mutants are with NHE1 protein terminated at amino acid, 321, 449, 543 and 735 as indicated. AP1 indicates mock transfected AP1 cells. Right arrows indicate the HA immunoreactive band of the 735, 543, 449 and 321 proteins. Right arrow with asterisk indicates non-specific immunoreactive band present in all cell lysates. Left arrow indicates HA immunoreactive band of WT NHE1 protein. C, Western blot of whole cell lysates of transiently transfected AP1 cells. Mutants are with NHE1 protein sequence terminated at amino acid, 321, 449, 543 and 735 as indicated. Right arrows indicate the HA immunoreactive bands as above. Left arrow with asterisk indicates non-specific immunoreactive band present in all cell lysates. D, Summary of levels of expression of stably expressed NHE1 protein in AP1 cells. Numbers are percent of the expression levels of mutants in comparison to wild type protein, mean ± S.E. n = at least 3 determinations.
Fig 2.
Surface localization of wild type (WT) and mutant NHE1 proteins in AP1 cells.
A, example of results and B, mean ± the S.E. n = at least 3 determinations. After cell surface biotinylation, equivalent amounts of a total cell lysate (T) and an unbound intracellular lysate (I) were examined by western blotting with anti-HA antibody to identify NHE1 protein. WT and mutant cell lines (321, 449, 543 and 735 as indicated) are cell lysates from cell lines stably expressing wild type NHE1 and mutant NHE1 proteins respectively. The % targeted to the cell surface is indicated.
Fig 3.
Analysis of Na+/H+ exchanger activity of wild type (WT) and mutant NHE1 proteins in AP1 cells.
Na+/H+ exchanger activity was assayed in stably transfected AP1 cells grown on coverslips as described above. A, Example of NHE1 activity of AP1 cells containing stably transfected wild type NHE1 and NHE1 mutant proteins. For clarity, only the recovery from ammonium chloride induced acidosis is shown for the mutant NHE1 proteins. NH4Cl, treatment with ammonium chloride. To induce acidosis following NH4Cl treatment there is a brief “Na Free” treatment. NaCl, = recovery period from acidosis in NaCl containing buffer. B, Summary of activity of WT and mutant (321, 449, 543 and 735 as indicated) NHE1 proteins in stably transfected AP1 cells. The NHE1 activity was measured after an ammonium chloride prepulse as illustrated in “A”. The initial rate of recovery in NaCl-containing medium was measured as ΔpH/s. Mutants activity are presented relative to that of the wild type protein, which was set at 100%. * indicates significantly different from wild type *P < 0.0001, n>8. The mean value of the wild type NHE1 activity was 0.026 ΔpH/s. C, Summary of Na+/H+ exchanger activity of NHE1-735 protein in comparison to wild type NHE1 protein. The 735-NHE1protein activity is displayed as a percent of the wild type (WT). 735c is the activity of the 735-NHE1 protein that has been corrected to the expression levels and surface targeting of the wild type NHE1 protein *P < 0.05, +P < 0.01, n>8. D, Resting pHi of wild type and 735-NHE1 protein containing cells at various times prior to and during ammonium chloride induced acidosis. pHrest, resting pHi prior to treatment with ammonium chloride; pHinitial, initial pHi after ammonium chloride treatment and prior to recovery in sodium containing medium; pHfinal, final pHi 3 minutes after recovery from ammonium chloride in sodium containing medium. n>8, ***P < 0.001. E, Characterization of Na+/H+ exchanger activity of wild type and 735-NHE1protein over different intracellular pHs (pHi). Cells containing wild type or 735-NHE1 protein were acidified to different levels by addition of varying amounts of ammonium chloride as described in the “Materials and Methods”. The initial rate of recovery was measured and recorded and Δ pH/s.
Fig 4.
Examination of mRNA and protein stability of mutant and wild type Na+/H+ exchangers.
A. mRNA levels of wild type and mutant NHE1 proteins 8 hours after treatment with 1.25 μM actinomycin D. mRNA levels were measured by quantitative RT-PCR. Levels are relative to the initial starting mRNA level prior to treatment and were corrected by the level of GAPDH. Results are mean +/- SE of at least 3 experiments. Individual experiments had 8 replicates. B. Protein levels of wild type and mutant NHE1 measured at time 0, (starting time) and up to 8 hours after cyclohexamide (50 μM) addition. Equal amounts of total proteins were loaded in each lane. NHE1 levels were determined by western blotting against the HA tag on the protein. Quantification was via were estimated using Image J 1.35 software. Insets are example western blots showing NHE1 levels (upper panel) in comparison to tubulin levels. Full sized NHE1 protein is indicated with an hour tubulin is indicated with an asterisk. Time points at 0 hr, 2, 4 and 8 are indicated. Results are mean +/- SE of at least 3 experiments.
Fig 5.
Analysis of NHE1 localization.
A, Localization of wild type and mutant NHE1 proteins in AP1 cells. Wild type (full length) and mutant NHE1 proteins were expressed in AP1 cells and examined for localization using antibodies against the HA-tag present on each protein. Left column shows the staining for NHE1 protein of the wild type or mutant NHE1 protein. Centre column shows DAPI staining and the right column shows merger of the two images. B, Effect of co-expression of wild type and mutant NHE1 proteins on protein targeting. All experiments in this Fig were using CHO cells which posses their own, endogenous NHE1 protein. DAPI, column 1, DAPI staining. NHE1, column 2, staining with monoclonal anti NHE1 antibody against the distal end of the C-terminal tail. Second antibody was coupled to Alex 488. HA-tag, column 3, staining with polyclonal antibody against the HA-tag present on NHE1 proteins transfected into cells. Second antibody was coupled to Alexa 647. Merge, column 4, merger of columns 1–3. Endogenous NHE1, row 1, CHO cells were not transfected with plasmid expressing NHE1 protein but possess their own endogenous NHE1 protein. 735, row 2, CHO cells containing their own endogenous NHE1 protein, were transfected with the 735-NHE1 protein. 321, row 3, CHO cells containing their own endogenous NHE1 protein, were transfected with the 321-NHE1 protein.