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Adiv Johnson, Yong Lee, Kuai Yu, Criss Hartzell, Lihua Marmorstein, Alan Marmorstein; Best Vitelliform Macular Dystrophy Mutants Oligomerize with Wild Type Bestrophin-1 and Exhibit Differential Effects on Protein Trafficking. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6080.
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© ARVO (1962-2015); The Authors (2016-present)
To determine how disease-causing mutations in the gene BEST1 alter function of its encoded protein, human Bestrophin-1 (hBest1). Since BEST1 mutants cause Best vitelliform macular dystrophy (BVMD) and mislocalization of channel proteins underlies the pathogenesis of other channelopathies, we investigated trafficking and co-trafficking of three hBest1 mutants (R218C, W93C, and V9M) causal for BVMD.
hBest1 mutants were expressed in MDCK cells, fhRPE cells, and in the RPE of mouse eyes using replication-defective adenoviruses. Localization was analyzed using immunofluorescence and confocal microscopy. Formation of oligomers between mutant and WT Best1 was investigated via FRET and co-immunoprecipitation in MDCK cells. Electrophysiology of WT and mutant hBest1 was studied using whole-cell patch clamp in transfected HEK-293 cells.
While WT hBest1 and R218C polarized to the basolateral plasma membrane of MDCK cells, both V9M and W93C were intracellular. In fhRPE cells, which express endogenous hBest1, R218C and W93C were properly localized, suggesting that WT hBest1 could rescue W93C mislocalization. FRET and co-immunoprecipitation experiments show that all three mutants oligomerize with WT hBest1 and that V9M can form oligomers with mouse Best1. In contrast to W93C, however, V9M remained intracellular in fhRPE cells and was mislocalized in the RPE of mouse eyes. Whole cell patch clamp recordings find that V9M impairs hBest1 Cl- conductance and diminishes the conductance of WT hBest1 when co-expressed. Co-infection of similar amounts of CFP-tagged hBest1 and YFP-tagged V9M in MDCK cells resulted in intracellular accumulation of both proteins.
We find that various BVMD-causing mutants exhibit differential effects on hBest1 localization and that the R218C, W93C, and V9M mutants can form oligomers with WT Best1. While the mutants R218C and W93C are properly localized to the basolateral plasma membrane of cells when expressed alone (R218C) or in combination (R218C, W93C) with WT hBest1, V9M does not traffic properly and is not rescued by the presence of WT Best1. Furthermore, we demonstrate that V9M can cause mislocalization of WT hBest1. We conclude that some mutations causal for BVMD disrupt the localization of Best1, which may underlie the loss of anion channel activity for that group of Best1 mutants.
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