Functional rescue of a kidney anion exchanger 1 trafficking mutant in renal epithelial cells.

Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1) can cause distal renal tubular acidosis (dRTA), a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK) cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients

DMSO can partially rescue the function of G701D kAE1.

<p>A, MDCK cells expressing WT or kAE1 mutants were grown on glass coverslips and either kept in control conditions or treated for 16 hours with DMSO, glycerol or low temperature. After incubation with 10 µM of the pH sensitive fluorescent probe BCECF-AM, coverslips were placed in a cuvette and perfused with Ringer’s solution containing sodium chloride at room temperature. The buffer was then switched to a chloride-free Ringer’s solution and the increase of BCECF fluorescence recorded. After completion of the experiment, the BCECF fluorescence was calibrated using high potassium buffers containing nigericin at three different pHs. The variation in pH units per min was calculated for each mutant in control or treated conditions. Values are mean ± SEM, n = 3 independent experiments. Asterisk indicates statistically significant difference with control condition (P≤0.05), calculated by one-way ANOVA. B, Cell lysates from MDCK cells expressing kAE1 WT or dRTA mutants and treated with (right panel) and without (left panel) DMSO for 16 hours, were incubated with SITS-Affi-Gel with and without incubation with the free inhibitor H₂DIDS. After three washes, the proteins were eluted and analyzed by immunoblot using a mouse anti-HA antibody followed by anti-mouse antibody coupled to HRP. T, total cell lysate, B, eluate from SITS-Affi-Gel, D, eluate from SITS-Affi-gel with pre-incubation with free H₂DIDS. Filled circles correspond to kAE1 proteins carrying complex oligosaccharide, open circles indicate kAE1 proteins carrying high mannose oligosaccharide.</p

The molecular chaperone VX-809 rescues trafficking but not function of G701D kAE1.

<p>A, MDCK cells expressing WT or kAE1 mutant were either kept in control conditions or treated with 0.5 or 1% DMSO for 16 hours prior to lysis. kAE1 proteins present in the lysate were resolved by immunoblot using a mouse anti-HA antibody followed by an anti-mouse antibody coupled to HRP. Glyceraldehyde phosphate dehydrogenase (GAPDH) was detected as a loading control. Filled circles correspond to kAE1 proteins carrying complex oligosaccharide, open circles indicate kAE1 proteins carrying high mannose oligosaccharide. B, MDCK cells expressing WT or kAE1 mutant were either kept in control conditions or treated with 3 µM of the molecular chaperone VX-809 for 24 hours prior to fixation. We then performed a two-step immunostaining as follows: cells were blocked and stained with a mouse anti-HA antibody followed by staining with an Alexa 488 coupled secondary antibody. Cells were then permeabilized and after blocking, stained again with a mouse anti-HA antibody followed by a Cy3 coupled secondary antibody. The samples were examined with a WaveFx spinning disk using a 60×oil immersion objective. The size bar corresponds to 10 µm. C, MDCK cells expressing WT or G701D kAE1 were grown on glass coverslips and either kept in control conditions or treated for 16 hours with 3 µM VX-809. After incubation with 10 µM of the pH sensitive fluorescent probe BCECF-AM, coverslips were placed in a PTI cuvette and perfused with a Ringer’s solution containing sodium chloride. The buffer was then switched to a chloride-free Ringer’s solution and the increase of BCECF fluorescence recorded. After completion of the experiment, the BCECF fluorescence was calibrated using high potassium buffers containing nigericin at three different pHs. The variation of pH units per min was calculated for each mutant in control or treated conditions. Values are mean ± SEM, n = 3 independent experiments. Asterisk indicates statistically significant difference with control condition (P≤0.05), calculated by one-way ANOVA. Dagger signs indicate significant difference (P≤0.05), compared with MDCK in control conditions.</p

Both DMSO and VX-809 increase synthesis of the G701D kAE1.

<p>A, MDCK cells expressing WT or mutant kAE1 were either kept in control conditions or treated for 4 hours with CHX (10 µg/ml), 1% DMSO (top panels) or 3 µM VX-809 (bottom panels) in presence or absence of CHX prior to lysis. After the protein concentration was determined, proteins (9 µg and 6 µg for DMSO and VX-809 treatments, respectively) were resolved by SDS-PAGE then immunobloted and detected using a mouse anti-HA antibody followed by an anti-mouse antibody coupled to HRP. Filled circles correspond to kAE1 proteins carrying complex oligosaccharide, open circles indicate kAE1 proteins carrying high mannose oligosaccharide. B, Histogram showing the percentage of WT or G701D kAE1 proteins present in lysates from cells kept in control conditions or treated as indicated. Values are mean ± SEM, n = 3 independent experiments. N.s. indicates non-statistically significant differences.</p

Low temperature incubations and chemical chaperones can partially rescue trafficking of dRTA mutants.

<p>MDCK cells expressing kAE1 WT or mutants were grown on semi-permeable filters until polarized, then treated either in control conditions, with 1% DMSO, 1% glycerol or at 30°C for 16 hours, fixed, blocked and stained with a mouse anti-HA antibody followed by an anti-mouse antibody coupled to Cy3 (red) fluorophore. Cells were then permeabilized, blocked and incubated with mouse anti-HA antibody again followed by an anti-mouse antibody coupled to Alexa 488 fluorophore (green). Yellow staining corresponds to overlap between green and red staining. The size bar represents 10 µm. The samples were examined with a spinning disk microscope using a 60×oil immersion objective. The images displayed are representative of 3 independent experiments.</p