Structural Characterization of SLC4A11, a Membrane Protein Mutated in Some Corneal Dystrophies

GONZALO L. VILAS, PATRICIO E. MORGAN, SAMPATH K. LOGANATHAN, ANITA QUON, AND JOSEPH R. CASEY

BAnff

Congreso; CSBMCB Annual Meeting on Membrane Proteins in Health and Disease; 2010

Canadian Society for Biochemistry, Molecular and Cellular Biology

The group of Descemet Membrane and Endothelial Dystrophies (DED) includes autosomal congenital hereditary endothelial corneal dystrophy (CHED2), corneal dystrophy and perceptive deafness (CDPD, Harboyan syndrome) and Fuchs corneal dystrophy (FECD).  Mutations in the SLC4A11 gene have been identified in patients with recessive CHED2, CDPD and late-onset FECD. SLC4A11 belongs to the SLC4 family of bicarbonate transporter proteins and is reported to function as an electrogenic sodium-borate co-transporter. To understand the normal function of the protein and the possible effects that pathogenic mutations may have on the etiology of corneal dystrophies, we studied structural and biochemical properties of SLC4A11 protein. SLC4A11 bound the anion exchanger inhibitor SITS with a significantly higher half maximal effective concentration than SLC4A1 (AE1), suggesting that stilbene disulfonates inhibit SLC4A11 activity. Despite SLC4A11 relatively low sequence identity with AE1 both proteins have a similar hydrophobicity profile, suggesting a similar membrane topology and structure. We propose that SLC4A11 is structured in a similar way as AE1. We tested this model by performing immunoflurescence studies and limited trypsinolysis of epitope-tagged versions of SLC4A11. These studies showed that both N- and C-termini of SLC4A11 are cytosolic and partially confirmed the orientation of some of the protein’s loops, confirming the proposed topological model SLC4A11. In vivo crosslinking experiments, native PAGE and immunoprecipitation showed that SLC4A11 exists as a dimer in the plasma membrane of HEK 293 cells. Finally, co-expression of wild type with disease-causing SLC4A11 mutants rescued the intracellularly-retained phenotype of the latter, providing a possible explanation for the genotypic nature of SLC4A11-related corneal dystrophies.