Stable dimeric assembly of the second membrane-spanning domain of CFTR (cystic fibrosis transmembrane conductance regulator) reconstitutes a chloride-selective pore.
Structural information is required to define the molecular basis for chloride conduction through CFTR (cystic fibrosis transmembrane conductance regulator). Towards this goal, we expressed MSD2, the second of the two MSDs (membrane-spanning domains) of CFTR, encompassing residues 857-1158 in Sf9 cells using the baculovirus system. In Sf9 plasma membranes, MSD2 migrates as expected for a dimer in non-dissociative PAGE, and confers the appearance of an anion permeation pathway suggesting that dimeric MSD2 mediates anion flux. To assess directly the function and quaternary structure of MSD2, we purified it from Sf9 cells by virtue of its polyhistidine tag and nickel affinity. Reconstitution of MSD2 into liposomes conferred a 4,4'-di-isothiocyanostilbene-2,2'-disulphonate-inhibitable, chloride-selective electrodiffusion pathway. Further, this activity is probably mediated directly by MSD2 as reaction of its single cysteine residue (Cys866) with the thiol modifying reagent, N(alpha)(3-maleimidylpropionyl)biocytin, inhibited chloride flux. Only MSD2 dimers were labelled by N(alpha)(3-maleimidylpropionyl)biocytin, supporting the idea that only dimeric MSD2 can mediate anion flux. As a further test of this hypothesis, we conducted a second purification procedure, wherein purified dimeric and monomeric MSD2 proteins were reconstituted separately. Only proteoliposomes containing stable MSD2 dimers mediated chloride electrodiffusion, providing direct evidence that dimeric MSD2 mediates chloride channel function. In summary, we have shown that the second membrane domain of CFTR can be purified and functionally reconstituted as a chloride channel, providing a tool for probing the structural basis of chloride conduction through CFTR.
Ramjeesingh
Mohabir
M
Programme in Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada M5G 1X8.
Ugwu
Francisca
F
Li
Canhui
C
Dhani
Sonja
S
Huan
Ling Jun
LJ
Wang
Yanchun
Y
Bear
Christine E
CE
D000818:Animals; D000838:Anions / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange / Q000379:methods; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004058:Diffusion; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera
D000838:Anions; D018118:Chloride Channels; D008081:Liposomes; D008565:Membrane Proteins; D010743:Phospholipids; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003545:Cysteine