title: A biochemical framework for SLC4A11, the plasma membrane protein defective in corneal dystrophies.
abstract: Mutations in the SLC4A11 protein, reported as a sodium-coup-led 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 H(2)DIDS, with a significantly higher half-maximal effective concentration than AE1. We conclude that stilbenedisulfonates interact with SLC4A11 but with a lower affinity than other SLC4 proteins. Disease-causing mutants divided into two classes on the basis of the half-maximal [H(2)DIDS] required for resin displacement and the fraction of protein binding H(2)DIDS, likely representing mildly misfolded and grossly misfolded proteins. Disease-causing SLC4A11 mutants are retained in the endoplasmic reticulum of HEK 293 cells. This phenotype could be partially rescued in some cases by growing the cells at 30 °C.
authors.affiliation: Membrane Protein Disease Research Group, Department of Physiology, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Canada T6G 2H7.
authors.forename: Gonzalo L
authors.initials: GL
authors.lastname: Vilas
authors.forename: Patricio E
authors.initials: PE
authors.lastname: Morgan
authors.forename: Sampath K
authors.initials: SK
authors.lastname: Loganathan
authors.forename: Anita
authors.initials: A
authors.lastname: Quon
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Casey
mesh_terms: D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000378:metabolism / Q000494:pharmacology; D000483:Alleles; D000595:Amino Acid Sequence; D027321:Anion Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017920:Antiporters / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts; D002462:Cell Membrane / Q000378:metabolism; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
chemical_list: D027321:Anion Transport Proteins; D017920:Antiporters; D002457:Cell Extracts; D000939:Epitopes; C425809:SLC4A11 protein, human; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D014357:Trypsin