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Front Physiol
2015 Jun 09;6:179. doi: 10.3389/fphys.2015.00179.
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Congenital chloride-losing diarrhea in a Mexican child with the novel homozygous SLC26A3 mutation G393W.
Reimold FR
,
Balasubramanian S
,
Doroquez DB
,
Shmukler BE
,
Zsengeller ZK
,
Saslowsky D
,
Thiagarajah JR
,
Stillman IE
,
Lencer WI
,
Wu BL
,
Villalpando-Carrion S
,
Alper SL
.
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Congenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl(-)/HCO(-) 3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting.
Figure 1. The novel SLC26A3 mutation G393W in a child with chloride-losing diarrhea. (A) Genomic DNA Sequence phoretograms of proband and parents. (B) Topographic model of hSLC26A3 (reproduced from Wedenoja et al., 2011) showing the predicted location of G393 within the transmembrane domain. (C) Alignment of mammalian SLC26A3 polypeptide sequences in the region of hSLC26A3 G393 (black box), showing partial conservation among species orthologs, with substitutions restricted to Ala and Ser (Polyphen-2 multiple sequence alignment).
Figure 2. SLC26A3 mutant G393W expressed in Xenopus oocytes exhibits modest loss-of-function. (A)
36Cl− influx into (n) uninjected oocytes (uninj.) or oocytes previously injected with 10 ng cRNA encoding hSLC26A3 WT (A3 WT) or its mutant hSLC26A3 G393W (A3 G393W). WT and mutant did not differ as judged by One-Way ANOVA analysis, but transport by G393W-expressing oocytes was significantly reduced as judged by Student's T-Test (p < 0.001). (B)
36Cl− efflux traces of representative individual oocytes previously uninjected (Uninj, open circles) or injected with 10 ng cRNA encoding hSLC26A3 WT (A3 WT, black squares) or mutant hSLC26A3 G393W (A3 G393W, open triangles), during sequential exposures to baths of sodium cyclamate (Na cyc.), ND-96, and Na cyc. (C) Summarized 36Cl− efflux rate constant data from (n) oocytes subjected to the protocol presented in (B). Values are means ± s.e.m. *p < 0.05 vs. hSLC26A3 WT (One-Way ANOVA). (D) Summarized 36Cl− efflux rate constant data from (n) uninjected oocytes (Uninj.) or oocytes previously injected with 10 ng of cRNA encoding hSLC26A3 WT or mutant G393W exposed first to 24 mM NaHCO3 plus 72 mM Na cyc (Cl−/HCO−3 exchange), followed by 96 mM Na cyc. 36Cl− efflux into HCO−3 solution, corrected for that into HCO−3-free cyclamate solution, did not differ statistically among groups.
Figure 3. SLC26A3 localization in Xenopus oocytes. Median intensity examples of confocal immunofluorescence images of Xenopus oocytes previously injected with 10 ng cRNA encoding (A) N-terminally HA-tagged wildtype hSLC263, (B) HA-tagged hSLC26A3 mutant G393W, or (C) oocytes previously uninjected with cRNA. (D) Mean values (± s.e.m.) of normalized fluorescent intensity (FI) for (n) oocytes similar to those presented in (A–C). FI did not differ between WT and mutant hSLC26A3-expressing oocytes, but both differed from the uninjected group (*p < 0.05).
Figure 4. SLC26A3 localization in HEK-293 cells. HEK-293 cells 48 h after transient transfection with cDNA encoding HA-SLC26A3 wildtype (A,B) or mutant HA-SLC26A3 G393 (C,D). HA epitope is red, and nuclei are stained with DAPI. (E) Immunoblot of whole cell RIPA lysates from HEK-293 cells prepared 24 h post-transient transfection with cDNA encoding HA-SLC26A3 wildtype (left two lanes) or mutant HA-SLC26A3 G393W (right two lanes). Actin immunoblot serves as load controls (below).
Figure 5. Localization of wildtype and mutant SLC26A3 polypeptides in nonconfluent, glass-grown Caco-2 cells. HA-SLC26A3 WT (upper panels) and HA-SLC26A3 G393G in subconfluent Caco-2 cells on glass, 24 h post-transient transfection, Cells were stained as indicated with anti-HA (left) and with anti-E-cadherin (middle). Merged images shown at right. Scale bar, 10 μm.
Figure 6. Localization of wildtype and mutant SLC26A3 polypeptides in confluent, filter-grown Caco-2 cells. HA-SLC26A3 WT (upper panels) and HA-SLC26A3 G393G (lower panels) in confluent Caco-2 cell monolayers grown on Transwell filters, 72 h post-transient transfection. Cells were stained as indicated with anti-HA, anti-α1-Na,K-ATPase, and phalloidin to detect F-actin. Merged images (with F-actin pseudocolored) shown at right. Scale bar, 10 μm.
Figure 7. Localization of wildtype SLC26A3 in filter-grown MDCK cells. Confluent, filter-grown, MDCK monolayers 48 h after transient transfection with cDNA encoding wildtype HA-SLC26A3. Fixed, permeabilized cells were costained with anti-HA (red) and antibody to the endogenous basolateral marker Na+,K+-ATPase (A, green) or to the endogenous apical marker gp135/podocalyxin (B, green). The x-z images at top are sections situated at the horizontal lines in the lower x-y panels. The x-y panels at bottom are sections situated at the blue horizontal lines in the upper x-z panels. Scale bars, 5 μm.
Figure 8. Localization of wildtype and mutant SLC26A3 polypeptides in confluent, glass-grown MDCK cells. X-Z confocal sections (A,C) and 20°-tilted Z stack of X-Y projections (B,D) of confluent glass-grown MDCK monolayers HA-SLC26A3-WT (left panels) and mutant HA-SLC26A3 G393W (Right panels), 72 h post-transient transfection. Anti-HA is shown in green, nuclear stain DRAQ5 in red. Scale bar, 10 μm.
Figure 9. Localization of wildtype and mutant SLC26A3 in glass-grown MDCK cells. Confluent, glass-grown MDCK monolayers 48 h after transient transfection with (A) cDNA encoding wildtype HA-SLC26A3 or with (B–D) cDNA encoding mutant HA-SLC26A3 G393W. Fixed, permeabilized cells were costained with anti-HA (red) and antibody to Na+,K+-ATPase (red). Nuclei are stained with DAPI. Location of x-z sections with respect to x-y sections is as described for Figure 6. Scale bar 5 μm.
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