Quaternary structure of the chloride channel ClC-2.
The chloride channel ClC-2 is thought to be essential for chloride homeostasis in neurons and critical for chloride secretion by the developing respiratory tract. In the present work, we investigated the quaternary structure of ClC-2 required to mediate chloride conduction. We found using chemical cross-linking and a novel PAGE system that tagged ClC-2 expressed in Sf9 cells exists as oligomers. Fusion of membranes from Sf9 cells expressing this protein confers double-barreled channel activity, with each pore exhibiting a unitary conductance of 32 pS. Polyhistidine-tagged ClC-2 from Sf9 cells can be purified as monomers, dimers, and tetramers. Purified, reconstituted ClC-2 monomers do not possess channel function whereas both purified ClC-2 dimers and tetramers do mediate chloride flux. In planar bilayers, reconstitution of dimeric ClC-2 leads to the appearance of a single, anion selective 32 pS pore, and tetrameric ClC-2 confers double-barreled channel activity similar to that observed in Sf9 membranes. These reconstitution studies suggest that a ClC-2 dimer is the minimum functional structure and that ClC-2 tetramers likely mediate double-barreled channel function.
Ramjeesingh
M
M
Research Institute in the Hospital for Sick Children, Toronto, Canada.
Li
C
C
Huan
L J
LJ
Garami
E
E
Wang
Y
Y
Bear
C E
CE
D000818:Animals; D000090007:CLC-2 Chloride Channels; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D002712:Chlorides / Q000493:pharmacokinetics*; D019281:Dimerization; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D020836:Protein Structure, Quaternary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D018411:Spodoptera / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D011994:Recombinant Proteins