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.

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

“…Phase b acidification is generally attributed to a finite permeability to NH 4 ϩ , and consequent phase c acidification is a H ϩ loading effect from the finite NH 4 ϩ permeability in phase b (17). The extents of acidification during phases b and c were both positively associated with the SLC4A11 expression level (40). The bar graph on the right shows the density quantification of HA-SLC4A11 normalized to ␣-tubulin.…”

“…It is well known that cornea and inner ear function depends on tightly controlled fluid and ion transport. At least 60 mutations have been identified as being associated with corneal endothelial dystrophy and/or perceptive deafness (40). A recent clinical study suggests that homozygous SLC4A11 mutated congenital hereditary endothelial dystrophy patients progress to Harboyan syndrome at a later age (perceptive deafness and corneal endothelial dystrophy (41)), whereas another study associated SLC4A11 mutants with the corneal ectasia disorder keratoconus (42).…”

SLC4A11 is an EIPA-sensitive Na+ permeable pHi regulator

“…Interestingly, SLC4A11 has low identity (14 -20%) with other SLC4 members. Phylogenetic analysis indicates that it does not cluster strongly with any of the three SLC4 functional groups (23). Lastly, SLC4A11 is postulated to be the mammalian homologue of the Arabidopsis thaliana borate transporter BOR1 (20).…”

High Throughput Assay Identifies Glafenine as a Corrector for the Folding Defect in Corneal Dystrophy–Causing Mutants of SLC4A11

“…Positions for epitope insertion were selected on the basis of proteolytic accessibility of the extracellular loops. 54 Double hemagglutinin epitope (HA) tags were inserted into extracellular loop 3, following amino acid R530 or P564 (Fig. 1A).…”

“…A269V was chosen because culture of this mutant in cells at 308C induced partial rescue to the cell surface. 54 E143K was chosen because dimerization with monomers that traffic to the plasma membrane resulted in a heterodimer at the plasma membrane. 27 These strategies suggest that these proteins have the potential to be cell-surface rescued by small molecules.…”