Structure-function analysis of the bestrophin family of anion channels.
The bestrophins are a newly described family of anion channels unrelated in primary sequence to any previously characterized channel proteins. The human genome codes for four bestrophins, each of which confers a distinctive plasma membrane conductance on transfected 293 cells. Extracellular treatment with methanethiosulfonate ethyltrimethylammonium (MTSET) of a series of substitution mutants that eliminate one or more cysteines from human bestrophin1 demonstrates that cysteine 69 is the single endogenous cysteine responsible for MTSET inhibition of whole-cell current. Cysteines introduced between positions 78-99 and 223-226 are also accessible to external MTSET, with MTSET modification at positions 79, 80, 83, and 90 producing a 2-6-fold increase in whole-cell current. The latter set of four cysteine-substitution mutants define a region that appears to mediate allosteric control of channel activity. Mapping of transmembrane topography by insertion of N-linked glycosylation sites and tobacco etch virus protease cleavage sites provides evidence for cytosolic N and C termini and an unexpected transmembrane topography with at least three extracellular loops that include positions 60-63, 212-227, and 261-267. These experiments provide the first structural analysis of the bestrophin channel family.
Tsunenari
Takashi
T
Department of Neuroscience,The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Sun
Hui
H
Williams
John
J
Cahill
Hugh
H
Smallwood
Philip
P
Yau
King-Wai
KW
Nathans
Jeremy
J
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D000075002:Bestrophins; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004594:Electrophysiology; D005136:Eye Proteins / Q000737:chemistry / Q000502:physiology*; D016678:Genome; D006031:Glycosylation; D006801:Humans; D007473:Ion Channels; D008698:Mesylates / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014981:Xenopus
D000838:Anions; C113133:BEST1 protein, human; C491856:Best1 protein, mouse; D000075002:Bestrophins; D018118:Chloride Channels; D005136:Eye Proteins; D007473:Ion Channels; D008698:Mesylates; D013439:Sulfhydryl Reagents; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D003545:Cysteine