TY - JOUR
T1 - Structure-Function Analysis of the Bestrophin Family of Anion Channels
AU - Tsunenari, Takashi
AU - Sun, Hui
AU - Williams, John
AU - Cahill, Hugh
AU - Smallwood, Philip
AU - Yau, King Wai
AU - Nathans, Jeremy
PY - 2003/10/17
Y1 - 2003/10/17
N2 - 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 methane-thiosulfonate ethyltrimethylammonium (MTSET) of a series of substitution mutants that eliminate one or more cysteines from human bestrophinl 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.
AB - 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 methane-thiosulfonate ethyltrimethylammonium (MTSET) of a series of substitution mutants that eliminate one or more cysteines from human bestrophinl 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.
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U2 - 10.1074/jbc.M306150200
DO - 10.1074/jbc.M306150200
M3 - Article
C2 - 12907679
AN - SCOPUS:0142071743
SN - 0021-9258
VL - 278
SP - 41114
EP - 41125
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 42
ER -