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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.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.

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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Superdex gel filtration. Purified EriC (230 μg) was applied to a Superdex FPLC column, and elution was monitored by 280-nm absorbance. A dashed line is drawn under the EriC peak; arrows mark the peak positions of a void volume marker (blue dextran), KcsA (74 kD), serum albumin (67 kD), cytochrome c (13 kD), and an included volume marker (imidazole).
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Figure 7: Superdex gel filtration. Purified EriC (230 μg) was applied to a Superdex FPLC column, and elution was monitored by 280-nm absorbance. A dashed line is drawn under the EriC peak; arrows mark the peak positions of a void volume marker (blue dextran), KcsA (74 kD), serum albumin (67 kD), cytochrome c (13 kD), and an included volume marker (imidazole).

Mentions: To complement the cross-linking experiments, we analyzed EriC by gel filtration chromatography and compared its migration to a reference membrane protein of known size, the K+ channel KcsA, a 74-kD homotetramer (Heginbotham et al. 1997). EriC runs slightly ahead of KcsA on a Superdex gel filtration column (Fig. 7). This result suggests that the EriC channel is substantially larger than its monomer molecular mass of 51 kD.


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)

Superdex gel filtration. Purified EriC (230 μg) was applied to a Superdex FPLC column, and elution was monitored by 280-nm absorbance. A dashed line is drawn under the EriC peak; arrows mark the peak positions of a void volume marker (blue dextran), KcsA (74 kD), serum albumin (67 kD), cytochrome c (13 kD), and an included volume marker (imidazole).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Superdex gel filtration. Purified EriC (230 μg) was applied to a Superdex FPLC column, and elution was monitored by 280-nm absorbance. A dashed line is drawn under the EriC peak; arrows mark the peak positions of a void volume marker (blue dextran), KcsA (74 kD), serum albumin (67 kD), cytochrome c (13 kD), and an included volume marker (imidazole).
Mentions: To complement the cross-linking experiments, we analyzed EriC by gel filtration chromatography and compared its migration to a reference membrane protein of known size, the K+ channel KcsA, a 74-kD homotetramer (Heginbotham et al. 1997). EriC runs slightly ahead of KcsA on a Superdex gel filtration column (Fig. 7). This result suggests that the EriC channel is substantially larger than its monomer molecular mass of 51 kD.

Bottom Line: ClC-type anion-selective channels are widespread throughout eukaryotic organisms.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.

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