Endogenous expression of mutant BEST1 results in decreased Cl^- and Ca²⁺ conductance in human retinal pigment epithelium.

Abstract

Purpose : Best disease is an incurable autosomal dominant form of macular degeneration caused by mutation of the BEST1 gene expressed in the retinal pigment epithelium (RPE). Best disease is characterized by accumulation of fluid and lipofuscin in the retina and a decreased light peak as measured by electro-oculogram. This study aimed to define the molecular consequences of mutant BEST1 within human pluripotent stem cell-derived RPE as a step towards finding an effective Best disease treatment.

Methods : We characterized a new human embryonic stem cell line, containing the disease-causing F305S BEST1 mutation, using standard assays including flow cytometry, PCR and teratoma formation. RPE generated from these mutant stem cells was analysed by immunofluorescence, Affymetrix microarray and electrophysiology in comparison to RPE generated from human embryonic stem cells with normal BEST1.

Results : The F305S mutant BEST1 stem cells display typical pluripotent cell morphology, express key pluripotency factors, and form teratomas containing endodermal, mesodermal and ectodermal cells. Affymetrix analysis revealed the diseased RPE has a similar transcriptional profile to normal RPE. Immunofluorescence showed the mutant RPE expresses RPE65 and ZO1 with expected staining patterns. The disease-specific RPE also expresses BEST1 protein. Consistent with published overexpression studies, the mutant BEST1 protein was localized in the cytoplasm. In contrast, BEST1 was localised at the plasma membrane in RPE generated from normal human embryonic stem cells. Whole cell patch clamping demonstrated no difference in Na⁺ or K⁺ conductance between normal and F305S mutant BEST1 RPE. However, the mutant RPE had a large and significant reduction in both Cl^- and Ca²⁺ conductances.

Conclusions : Our data are consistent with BEST1 acting as a Ca²⁺-activated Cl^- channel in human RPE, with both Cl^- and Ca²⁺ currents reduced by the disease-causing congenital F305S BEST1 mutation. The fact these data represent endogenous BEST1 activity, not artificial overexpression of exogenous BEST1, emphasizes the clinical relevance of our findings. Ongoing analyses are defining the pathways that link impaired Cl^- and Ca²⁺ homeostasis with key Best disease symptoms such as fluid and lipofuscin accumulation, and decreased electro-oculogram light peak. In turn, this information will provide new targets for Best disease treatment.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.