CLIC-1 functions as a chloride channel when expressed and purified from bacteria.
CLIC-1 is a member of a family of proteins related to the bovine intracellular chloride channel p64 which has been proposed to function as a chloride channel. We expressed CLIC-1 as a glutathione S-transferase fusion protein in bacteria. The fusion protein was purified by glutathione affinity, and CLIC-1 was released from its fusion partner by digestion with thrombin. After further purification, CLIC-1 was reconstituted into phospholipid vesicles by detergent dialysis. Chloride permeability of reconstituted vesicles was assessed using a valinomycin dependent chloride efflux assay, demonstrating increased vesicular chloride permeability with CLIC-1 compared with control. CLIC-1-dependent chloride permeability was inhibited by indanyloxyacetic acid-94 with an apparent IC(50) of 8.6 micrometer. The single channel properties of CLIC-1 were determined using the planar lipid bilayer technique. We found that CLIC-1 forms a voltage-dependent, Cl-selective channel with a rectifying current-voltage relationship and single channel conductances of 161 +/- 7.9 and 67.5 +/- 6.9 picosiemens in symmetric 300 and 150 mm KCl, respectively. The anion selectivity of this activity is Br approximately Cl > I. The open probability of CLIC-1 channels in planar bilayers was decreased by indanyloxyacetic acid-94 with an apparent IC(50) of 86 micrometer at 50 mV. These data convincingly demonstrate that CLIC-1 is capable of forming a novel, chloride-selective channel in the absence of other subunits or proteins.
Tulk
B M
BM
Department of Internal Medicine, St. Louis University, the Department of Cell Biology and Physiology, Washington University School of Medicine, and St. Louis Veterans Affairs Medical Center, St. Louis, Missouri 63106, USA.
Schlesinger
P H
PH
Kapadia
S A
SA
Edwards
J C
JC
D000818:Animals; D002417:Cattle; D018118:Chloride Channels / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics; D015640:Ion Channel Gating; D017136:Ion Transport; D008051:Lipid Bilayers; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018118:Chloride Channels; D002712:Chlorides; D008051:Lipid Bilayers; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase