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High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel.

Maduke M, Pheasant DJ, Miller C - J. Gen. Physiol. (1999)

Bottom Line: ClC-type anion-selective channels are widespread throughout eukaryotic organisms.BLAST homology searches reveal that many microbial genomes also contain members of the ClC family.Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02254-9110, USA.

ABSTRACT
ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli-derived ClC Cl(-) channel homologue, "EriC," the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.

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Glutaraldehyde cross-linking of EriC. EriC was treated with glutaraldehyde as in materials and methods, and the samples were run on a 10% polyacrylamide gel.(1) 6 μg EriC, no glutaraldehyde, (2–5) 6 μg EriC cross-linked for 1, 10, 20, or 60 min, respectively, (6) 6 μg EriC incubated with 1% SDS before reaction with glutaraldehyde for 60 min.
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Figure 6: Glutaraldehyde cross-linking of EriC. EriC was treated with glutaraldehyde as in materials and methods, and the samples were run on a 10% polyacrylamide gel.(1) 6 μg EriC, no glutaraldehyde, (2–5) 6 μg EriC cross-linked for 1, 10, 20, or 60 min, respectively, (6) 6 μg EriC incubated with 1% SDS before reaction with glutaraldehyde for 60 min.

Mentions: Glutaraldehyde, a nonspecific cross-linking agent, has been used convincingly to report the oligomeric state of several membrane proteins (Canals 1992; Craig 1982a,Craig 1982b; Rabon et al. 1990). Reaction of glutaraldehyde with EriC gives a clean result (Fig. 6). Within 10 min of reaction, the 38-kD band is quantitatively converted into a band migrating at ∼90 kD; no conversion to higher mass species occurs over the next 60 min. The possibility of intermolecular cross-linking is largely eliminated by the control experiment showing no cross-linking of EriC dispersed under denaturing conditions in SDS. In addition, variation of the protein-detergent ratio over two orders of magnitude failed to alter the cross-linking, a result that rules out artifactual dimerization resulting from multiple protein molecules sharing the same micelle (Fleming et al. 1997). Moreover, similar results were obtained with EriC reconstituted at very low density (0.09 μg/mg lipid), where most liposomes have either one EriC channel or none. Cross-linking was robust to varying glutaraldehyde or protein concentrations; while the rates of cross-linking increased with these variables, the overall pattern, particularly the absence of high aggregates, was unchanged.


High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel.

Maduke M, Pheasant DJ, Miller C - J. Gen. Physiol. (1999)

Glutaraldehyde cross-linking of EriC. EriC was treated with glutaraldehyde as in materials and methods, and the samples were run on a 10% polyacrylamide gel.(1) 6 μg EriC, no glutaraldehyde, (2–5) 6 μg EriC cross-linked for 1, 10, 20, or 60 min, respectively, (6) 6 μg EriC incubated with 1% SDS before reaction with glutaraldehyde for 60 min.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230540&req=5

Figure 6: Glutaraldehyde cross-linking of EriC. EriC was treated with glutaraldehyde as in materials and methods, and the samples were run on a 10% polyacrylamide gel.(1) 6 μg EriC, no glutaraldehyde, (2–5) 6 μg EriC cross-linked for 1, 10, 20, or 60 min, respectively, (6) 6 μg EriC incubated with 1% SDS before reaction with glutaraldehyde for 60 min.
Mentions: Glutaraldehyde, a nonspecific cross-linking agent, has been used convincingly to report the oligomeric state of several membrane proteins (Canals 1992; Craig 1982a,Craig 1982b; Rabon et al. 1990). Reaction of glutaraldehyde with EriC gives a clean result (Fig. 6). Within 10 min of reaction, the 38-kD band is quantitatively converted into a band migrating at ∼90 kD; no conversion to higher mass species occurs over the next 60 min. The possibility of intermolecular cross-linking is largely eliminated by the control experiment showing no cross-linking of EriC dispersed under denaturing conditions in SDS. In addition, variation of the protein-detergent ratio over two orders of magnitude failed to alter the cross-linking, a result that rules out artifactual dimerization resulting from multiple protein molecules sharing the same micelle (Fleming et al. 1997). Moreover, similar results were obtained with EriC reconstituted at very low density (0.09 μg/mg lipid), where most liposomes have either one EriC channel or none. Cross-linking was robust to varying glutaraldehyde or protein concentrations; while the rates of cross-linking increased with these variables, the overall pattern, particularly the absence of high aggregates, was unchanged.

Bottom Line: ClC-type anion-selective channels are widespread throughout eukaryotic organisms.BLAST homology searches reveal that many microbial genomes also contain members of the ClC family.Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02254-9110, USA.

ABSTRACT
ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli-derived ClC Cl(-) channel homologue, "EriC," the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.

Show MeSH
Related in: MedlinePlus