Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Abstract

SummaryBestrophin-1, an integral membrane protein encoded by the BEST1 gene, is localized predominantly to the basolateral membrane of the retinal pigment epithelium. Mutations in the BEST1 gene have been associated with Best vitelliforme macular dystrophy (BMD), a central retinopathy with autosomal dominant inheritance and variable penetrance. Over 120 disease-causing mutations are known, the majority of which result in amino acid substitutions within four mutational hot-spot regions in the highly conserved N-terminal half of the protein. Although initially thought to impair Cl -channel function, the molecular pathology of BEST1 mutations is still controversial. We have analyzed the subcellular localization of 13 disease-associated BEST1 mutant proteins in polarized MDCK II cells, an established model of apical to basolateral protein sorting. Immunostaining demonstrated that nine of the 13 mutant proteins failed to integrate into the cell membrane. The defective proteins were predominantly retained in the cytoplasm, whereas wild-type bestrophin-1 revealed cell membrane localization. Functional analysis of I -fluxes in HEK-293 cells showed that all mutants exhibited a significant reduction in anion conductance. Our data indicate that defective intracellular trafficking could be a common cause of BMD accompanied by impaired anion conductance, representing a loss of anion channel function that is probably due to mistargeting of mutant protein.

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

“…Upon hypotonic stimulation, the simultaneous activation of the endogenous LRRC8A-mediated VRAC current in HEK293 cells (6, 7) renders it impossible to distinguish endogenous VRACs from heterologously expressed BEST1 currents. To avoid these methodological difficulties, we resorted to Xenopus oocytes, a polarized cell system that provides an important precondition for plasma membrane localization of BEST1 (41). After manual defolliculation and careful monitoring of each single batch for endogenous swellingactivated whole-cell currents, we recorded BEST1-mediated swelling-sensitive currents that were significantly enlarged relative to endogenous I swell .…”

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

“…BEST1, the most studied paralog, was first identified by genetic linkage to a juvenile-onset macular degeneration disease called "Best disease" (6). Nearly 200 mutations in BEST1 have been associated with retinal degenerative disorders, and the vast majority of those that have been characterized cause Cl − channel dysfunction (7)(8)(9)(10)(11). Studies have also indicated that certain bestrophin channels are important for cell volume regulation (12)(13)(14).…”

Stoichiometry and specific assembly of Best ion channels

“…This coiled-coil domain has been identified as one of the four regions where mutations are associated with disease (42). Some of the mutations in the coiled-coil affect the surface expression of hBest1 in MDCKII cells (47), an expected outcome of disrupted assembly and a clear cause of pathology. However, at least one disease-causing mutation in the coiled coil (R218C) has no effect on the surface expression (47), suggesting that coiled-coil domain has other additional functions in the channel.…”

“…Some of the mutations in the coiled-coil affect the surface expression of hBest1 in MDCKII cells (47), an expected outcome of disrupted assembly and a clear cause of pathology. However, at least one disease-causing mutation in the coiled coil (R218C) has no effect on the surface expression (47), suggesting that coiled-coil domain has other additional functions in the channel. We find that transplantation of the coiled-coil domain confers the large current of hBest1 on to hBest3 but only when the coiled coil is accompanied by cotransplantation of Ca 2+ -binding domain of hBest1.…”

“…After 1-2 d of expression, oocytes were devitillinized and imaged using TIRFM as described in detail in SI Materials and Methods. Iodide imaging (43,47) is described in SI Materials and Methods.…”