Abstract
ABSTRACTAutosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca2+-gated Cl− channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl− ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl− conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1.
Highlights
Bestrophin-1 is a homopentameric channel protein primarily expressed in the basolateral membrane of the retinal pigment epithelium (RPE) (Marmorstein et al, 2000; Kane Dickson et al, 2014)
We previously showed that nine autosomal recessive bestrophinopathy (ARB)-associated bestrophin-1 mutant proteins have significantly reduced Cl− conductance compared with the WT protein, and provided evidence that the loss of function was associated with protein misfolding in the secretory pathway
We show that both WT and mutant ARB-associated bestrophin-1 proteins are subject to a degree of proteasomal degradation and show that the loss of function of the mutant proteins results from misfolding rather than having active site mutations
Summary
Bestrophin-1 is a homopentameric channel protein primarily expressed in the basolateral membrane of the retinal pigment epithelium (RPE) (Marmorstein et al, 2000; Kane Dickson et al, 2014). Exogenous expression of bestrophin-1 results in a novel Ca2+activated Cl− current (Sun et al, 2002); it is highly permeable to HCO3 (Qu and Hartzell, 2008) and has been reported to act as a glutamate channel in hippocampal astrocytes (Woo et al, 2012) These data, together with the crystal structure of bestrophin-1 orthologues, suggest that it is an anion channel (Kane Dickson et al, 2014). Most mutant proteins were mislocalised (7 of 9 examined), and had reduced stability resulting from proteasomal degradation (6 of 9 examined) (Burgess et al, 2008; Davidson et al, 2009, 2011) These ARBassociated mutations seem to disrupt bestrophin-1 function, at least in part, by disrupting the protein’s folding, leading to retention and degradation by the quality control systems that eliminate misfolded proteins from the secretory pathway (Christianson and Ye, 2014)
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