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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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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.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