Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1.
CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.
Warton
Kristina
K
Centre for Immunology, St Vincent's Hospital and University of New South Wales, Sydney NSW 2010, Australia.
Tonini
Raffaella
R
Fairlie
W Douglas
WD
Matthews
Jacqueline M
JM
Valenzuela
Stella M
SM
Qiu
Min Ru
MR
Wu
Wan Man
WM
Pankhurst
Susan
S
Bauskin
Asne R
AR
Harrop
Stephen J
SJ
Campbell
Terence J
TJ
Curmi
Paul M G
PM
Breit
Samuel N
SN
Mazzanti
Michele
M
D000818:Animals; D016466:CHO Cells; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D002942:Circular Dichroism; D006224:Cricetinae; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D018118:Chloride Channels; D002712:Chlorides; D008051:Lipid Bilayers; D008081:Liposomes; D011994:Recombinant Proteins