Abstract
Purpose :
A number of misfolding mutations in the ion-transporter SLC4A11 are associated with Fuchs' Endothelial Corneal Dystrophy (FECD). Inspired by CFTR misfolding, the non-steroidal anti-inflammatory (NSAID) glafenine, reportedly demonstrates some capacity to increase the surface expression of selected mutants. To evaluate the effect of glafenine and potentially related prostaglandin synthesis modulators in a quantitative manner, we developed a flow-cytometry-based assay for SLC4A11 folding correction.
Methods :
Plasmids containing wild-type SLC4A11, SLC4A11-G709E, and SLC4A11-E143K were obtained or cloned with an N-terminal hemagglutinin (HA) tag, a double HA-tag between residues 564 and 565, or a C-terminal GFP tag. HEK293 cells were transfected with these constructs and treated with glafenine, and a selection of prostaglandin pathway modulators at concentrations ranging from 10 nM to 20 µM, for between 12 and 48 hours. Surface expression of SLC4A11 was assessed by flow cytometry with an anti-HA or anti-GFP antibody.
Results :
For wild-type SLC4A11, approximately 35 % of GFP positive cells could be stained with an anti-GFP antibody. Surface expression of SLC4A11-G709E was not detected, and approximately 7 % of cells showed low levels of SLC4A11-E143K surface expression. Treatment of these SLC4A11-E143K cells with 10 µM glafenine increased expression to 10.4 %, while minimal changes were observed upon treatment of the more severe G709E mutant. Prostagladin synthesis inhibitors, which have shown success in treating CFTR misfolding, demonstrated similarly minimal effects. Somewhat unexpectedly, the N-terminal HA tag and C-terminal GFP tag could be stained without permeabilization – indicating their exposure at the cell surface.
Conclusions :
Previous reports of glafenine's capacity for folding correction analyzed small numbers of cells primarily through microscopy. That glafenine appears to be less effective when assessed at the population level may indicate a heterogeneous response in which only a subset of cells are amenable to folding correction. Understanding the cause will likely be essential to developing of non-surgical interventions for SLC4A11-linked FECD. Staining of the N and C-termini of SLC4A11 without permeabilization updates the long-standing homology model which posits both termini are located inside the cell.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.