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Abstract:

Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 ((\mathrm{SLC4A11}{WT})) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations ((\mathrm{SLC4A11}{MU})). (\mathrm{SLC4A11}{WT}) and (\mathrm{SLC4A11}{MU}) hCEnC lines were generated to express either SLC4A11 variant 2 ((V2{WT}) and (V2{MU})) or variant 3 ((V3{WT}) and (V3{MU})), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and (NH_3)-induced membrane conductance. We demonstrate (\mathrm{SLC4A11}^{-/-}) and (\mathrm{SLC4A11}{MU}) hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to (\mathrm{SLC4A11}{WT}) hCEnC. Additionally, (\mathrm{SLC4A11}^{-/-}) hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to (\mathrm{SLC4A11}{WT}) hCEnC. Induction with 10mM (NH_4Cl) led (\mathrm{SLC4A11}{WT}) hCEnC to depolarize; conversely, (\mathrm{SLC4A11}^{-/-}) hCEnC hyperpolarized and the majority of (\mathrm{SLC4A11}{MU}) hCEnC either hyperpolarized or had minimal membrane potential changes following (NH_4Cl) induction. Immunostaining of primary hCEnC and (\mathrm{SLC4A11}{WT}) hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.