Investigation of the functional impact of CHED- and FECD4-associated SLC4A11 mutations in human corneal endothelial cells

• Published in: PloS one Vol. 19; no. 1; p. e0296928
• Main Authors: Chung, Doug D., Chen, Angela C., Choo, Charlene H., Zhang, Wenlin, Williams, Dominic, Griffis, Christopher G., Bonezzi, Paul, Jatavallabhula, Kavya, Sampath, Alapakkam P., Aldave, Anthony J.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 22.01.2024; Public Library of Science (PLoS)
• Subjects: Amino acids; Ammonia; Ammonium chloride; Anion Transport Proteins - genetics; Antiporters - genetics; Biology and Life Sciences; Care and treatment; Cell migration; Cell surface; Cell viability; Cells; Chromosome Disorders; Cloning; Congenital heart disease; Cornea; Corneal Dystrophies, Hereditary - genetics; CRISPR; Diagnosis; Dystrophy; Edema; Endothelial Cells; Endothelium; Fuchs' Endothelial Dystrophy - genetics; Genetic aspects; Health aspects; Human subjects; Humans; Investigations; Invoices; Isoforms; Localization; Medicine and Health Sciences; Membrane conductance; Membrane potential; Membranes; Mutation; Oxidative stress; Penicillin; Proteins; Research and Analysis Methods; Scientific equipment and supplies industry; SLC4A Proteins; Substrates; India; United States
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0296928

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 (SLC4A11 WT ) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11 MU ). SLC4A11 WT and 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 SLC4A11 -/- and SLC4A11 MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11 WT hCEnC. Additionally, SLC4A11 -/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11 WT hCEnC. Induction with 10mM NH 4 Cl led SLC4A11 WT hCEnC to depolarize; conversely, SLC4A11 -/- hCEnC hyperpolarized and the majority of SLC4A11 MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH 4 Cl induction. Immunostaining of primary hCEnC and 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.