title: The anion-selective pore of the bestrophins, a family of chloride channels associated with retinal degeneration.
abstract: Mutations in human bestrophin-1 (VMD2) are genetically linked to a juvenile form of macular degeneration and autosomal dominant vitreoretinochoroidopathy. Recently, it has been proposed that bestrophins are Cl- channels and that the putative second transmembrane domain participates in forming the bestrophin pore. However, the structural determinants of Cl- ion permeation through the channel pore are not known. Here we systematically replaced every amino acid in mouse bestrophin-2 (mBest2) between positions 69 and 104 with cysteine. We then measured the effects on the relative permeability and conductance of the channel to Cl- and SCN- (thiocyanate) and determined the accessibility of the cysteine-substituted amino acids to extracellularly applied, membrane-impermeant sulfhydryl reagents. Unlike K+ channels, the amino acids forming the mBest2 selectivity filter are not discretely localized but are distributed over approximately 20 amino acids within the transmembrane domain. Cysteine-substituted amino acids in the selectivity filter are easily accessible to extracellularly applied sulfhydryl reagents and select for anionic sulfhydryl reagents over cationic ones. Understanding the structure of the anion conduction pathway of bestrophins provides insights into how mutations produce channel dysfunction and may provide important information for development of therapeutic strategies for treating macular degeneration.
authors.affiliation: Department of Cell Biology, Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Qu
authors.forename: Li-Ting
authors.initials: LT
authors.lastname: Chien
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Cui
authors.forename: H Criss
authors.initials: HC
authors.lastname: Hartzell
mesh_terms: D000595:Amino Acid Sequence / Q000502:physiology; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry / Q000494:pharmacology; D000075002:Bestrophins; D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary / Q000235:genetics; D012160:Retina / Q000166:cytology / Q000378:metabolism* / Q000503:physiopathology; D012162:Retinal Degeneration / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology
chemical_list: D000596:Amino Acids; C113133:BEST1 protein, human; D000075002:Bestrophins; D018118:Chloride Channels; D002712:Chlorides; D005136:Eye Proteins; D049452:Green Fluorescent Proteins; D003545:Cysteine