Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Invest Ophthalmol Vis Sci. 2018 Aug 1;59(10):4258-4267. doi: 10.1167/iovs.18-24301.
SLC4A11 is a plasma membrane protein of corneal endothelial cells. Some mutations of the SLC4A11 gene result in SLC4A11 protein misfolding and failure to mature to the plasma membrane. This gives rise to some cases of Fuchs' endothelial corneal dystrophy (FECD) and congenital hereditary endothelial dystrophy (CHED). We screened ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs) for their ability to correct SLC4A11 folding defects.
Five ophthalmic NSAIDs were tested for their therapeutic potential in some genetic corneal dystrophy patients. HEK293 cells expressing CHED and FECD-causing SLC4A11 mutants were grown on 96-well dishes in the absence or presence of NSAIDs. Ability of NSAIDs to correct mutant SLC4A11 cell-surface trafficking was assessed with a bioluminescence resonance energy transfer (BRET) assay and by confocal microscopy. The ability of mutant SLC4A11-expressing cells to mediate water flux (SLC4A11 mediates water flux across the corneal endothelial cell basolateral membrane as part of the endothelial water pump) was measured upon treatment with ophthalmic NSAIDs.
BRET-assays revealed significant rescue of SLC4A11 mutants to the cell surface by 4 of 5 NSAIDs tested. The NSAIDs, diclofenac and nepafenac, were effective in moving endoplasmic reticulum-retained missense mutant SLC4A11 to the cell surface, as measured by confocal immunofluorescence. Among intracellular-retained SLC4A11 mutants, 20 of 30 had significant restoration of cell surface abundance upon treatment with diclofenac. Diclofenac restored mutant SLC4A11 water flux activity to the level of wild-type SLC4A11 in some cases.
These results encourage testing diclofenac eye drops as a treatment for corneal dystrophy in patients whose disease is caused by some SLC4A11 missense mutations.
SLC4A11 是角膜内皮细胞的质膜蛋白。该基因的某些突变导致 SLC4A11 蛋白错误折叠和无法成熟到质膜。这导致了一些 Fuchs 内皮角膜营养不良（FECD）和先天性遗传性内皮营养不良（CHED）病例的发生。我们筛选了眼科非甾体抗炎药（NSAIDs），以评估它们纠正 SLC4A11 折叠缺陷的能力。
测试了五种眼科 NSAIDs 在一些遗传性角膜营养不良患者中的治疗潜力。在不存在或存在 NSAIDs 的情况下，将表达 CHED 和 FECD 致病 SLC4A11 突变体的 HEK293 细胞在 96 孔板上培养。通过生物发光共振能量转移（BRET）测定和共焦显微镜评估 NSAIDs 纠正突变 SLC4A11 细胞表面转运的能力。在用眼科 NSAIDs 处理后，测量表达突变 SLC4A11 的细胞介导水通量的能力（SLC4A11 作为内皮水泵的一部分介导水穿过角膜内皮细胞基底外侧膜的通量）。
BRET 测定显示，在所测试的 5 种 NSAIDs 中有 4 种显著挽救了 SLC4A11 突变体到细胞表面。NSAIDs 双氯芬酸和奈帕芬酸可有效将内质网滞留的错义突变 SLC4A11 转移到细胞表面，这通过共聚焦免疫荧光测定得以证实。在细胞内滞留的 SLC4A11 突变体中，有 20/30 种在使用双氯芬酸治疗后，细胞表面丰度显著恢复。在某些情况下，双氯芬酸可恢复突变 SLC4A11 的水通量活性至野生型 SLC4A11 的水平。
这些结果鼓励在某些 SLC4A11 错义突变引起疾病的患者中，测试双氯芬酸滴眼液作为角膜营养不良的治疗方法。
