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J. Biol. Chem., Vol. 275, Issue 35, 26986-26993, September 1, 2000
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From the 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 IC50 of 8.6 µM. 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
CLIC-1 Functions as a Chloride Channel When Expressed and
Purified from Bacteria*
,, and¶
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
Cl > I. The open
probability of CLIC-1 channels in planar bilayers was decreased by
indanyloxyacetic acid-94 with an apparent IC50 of 86 µM 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.
*
This work was supported by National Institutes of Health
Grants R29 DK46212 and RO1 AR44838 and grants from the Barnes-Jewish Hospital Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Renal Division
657/111-JC, St. Louis VA Medical Center, 915 N. Grand Blvd., St. Louis,
MO 63106. Tel.: 314-652-4100 (ext. 5302); Fax: 314-289-7012; E-mail:
John.Edwards3@med.va.gov.
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