Purchase this article with an account.
Adiv A Johnson, Lori A Bachman, Benjamin J Gilles, Lihua Y Marmorstein, Jose S Pulido, Alan D Marmorstein; Functional characterization of a novel, truncated bestrophin mutant in a young patient with compound heterozygous mutations in BEST1 and autosomal recessive bestrophinopathy. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4226.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Mutations in BEST1, encoding Bestrophin-1 (Best1), cause multiple retinal degenerative diseases, including autosomal recessive bestrophinopathy (ARB). Best1 is a pentameric anion channel expressed to the basolateral plasma membrane of the retinal pigment epithelium (RPE). We present a novel frameshift mutation, I366fsX382, found in a patient with ARB. She is compound heterozygous for this mutation as well as the previously reported mutation R141H. To gain insight into the pathogenesis of ARB, we characterized the effects of these mutations on Best1.
Using confocal microscopy and immunofluorescence, we assessed localization of wild-type (WT) and mutant Best1 after expressing tagged forms of Best1 in iPSC-derived RPE (dRPE) cells via adenovirus-mediated gene transfer. Oligomerization was evaluated by co-immunoprecipitation. Anion channel activity was assessed via whole-cell patch clamp analysis in transfected HEK293 cells.
We find that the truncation mutant I366fsX382 exhibits robust Cl- currents comparable to WT Best1 in transfected HEK293 cells. R141H exhibited diminished anion channel activity. In dRPE, which express endogenous Best1, overexpressed WT and R141H Best1 were properly localized. I366fsX382 was mislocalized to intracellular compartments. Overexpressed WT and R141H Best1 co-localized with endogenous Best1 in the plasma membrane while both I366fsX382 and endogenous Best1 were mislocalized. Overexpressed WT, R141H, and I366fsX382 Best1 all co-immunoprecipitated with endogenous Best1.
I366fsX382 exhibited robust channel activity and oligomerized with non-truncated Best1, indicating that the first 366 amino acids of Best1 are sufficient for anion channel activity and homo-oligomerization. I366fsX382 failed to localize to the basolateral plasma membrane when expressed in dRPE, demonstrating that the C-terminal domain of Best1 plays a role in protein trafficking. Endogenous Best1 was also mislocalized in cells overexpressing this mutant. This suggests that, in the heterozygous parent without disease, both mutant and WT Best1 are mislocalized. This indicates that mislocalization alone is not sufficient to cause ARB. These data enhance our understanding of the pathogenesis of ARB as well as the protein regions governing Best1’s trafficking, homo-oligomerization, and channel activity.
This PDF is available to Subscribers Only