Journal
BIOCHEMISTRY
Volume 50, Issue 12, Pages 2157-2169Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi101887z
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Funding
- Alberta Heritage Foundation for Medical Research
- Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET)
- Canadian Institutes of Health Research
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Mutations in the SLC4A11 protein, reported as a sodium-coupled borate transporter of the human plasma membrane, are responsible for three corneal dystrophies (CD): congenital hereditary endothelial dystrophy type 2, Harboyan syndrome, and late-onset Fuch's CD. To develop a rational basis to understand these diseases, whose point mutations are found throughout the SLC4A11 sequence, we analyzed the protein biochemically. Hydropathy analysis and an existing topology model for SLC4A1 (AE1), a bicarbonate transporter with the lowest evolutionary sequence divergence from SLC4A11, formed the basis to propose an SLC4A11 topology model. Immunofluorescence studies revealed the cytosolic orientation of N- and C-termini of SLC4A11. Limited trypsinolysis of SLC4A11 partially mapped the folding of the membrane and cytoplasmic domains of the protein. The binding of SLC4A11 to a stilbenedisulfonate inhibitor resin (SITS-Affi-Gel) was prevented by preincubation with H2DIDS, with a significantly higher half-maximal effective concentration than AE1. We conclude that stilbenedisulfonates interact with SLC4A1 1 but with a lower affinity than other SLC4 proteins. Disease-causing mutants divided into two classes on the basis of the half-maximal [H2DIDS] required for resin displacement and the fraction of protein binding H2DIDS, likely representing mildly misfolded and grossly misfolded proteins. Disease-causing SLC4A11 mutants are retained in the endoplasmic reticulum of FIEK 293 cells. This phenotype could be partially rescued in some cases by growing the cells at 30 degrees C.
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