RESUMO
Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl- channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy. Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp. Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1. Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration-approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.
Assuntos
Antineoplásicos/farmacologia , Bestrofinas/genética , Oftalmopatias Hereditárias/tratamento farmacológico , Regulação da Expressão Gênica/fisiologia , Glicolatos/farmacologia , Fenilbutiratos/farmacologia , Doenças Retinianas/tratamento farmacológico , Epitélio Pigmentado da Retina/efeitos dos fármacos , Western Blotting , Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Cicloeximida/farmacologia , Eletroforese em Gel de Poliacrilamida , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Genes Recessivos , Células HEK293/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , TransfecçãoRESUMO
The last decade has witnessed an exponential increase in interest in one of the great mysteries of nerve cell biology: Specifically, how do neurons know where to place the ion channels that control their excitability? Many of the most important insights have been gleaned from studies on the voltage-gated potassium channels (Kvs) which underlie the shape, duration and frequency of action potentials. In this review, we gather recent evidence on the expression, trafficking and maintenance mechanisms which control the surface density of Kvs in different subcellular compartments of neurons and how these may be regulated to control cell excitability.