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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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Related in: MedlinePlus

Velocity sedimentation of Eric. A280 in each sample cell is plotted as a function of radius from the axis of rotation. Each scan was made at a different time during the centrifugation run; the scans made at 27, 107, and 187 min are specifically labeled. Fits to the Fujita-MacCosham function yields values for the sedimentation coefficient, s, and diffusion coefficient, D, which in turn yield estimates of molar mass, Mw. (A) KcsA: s = 6.73 S, D = 6.55 × 10−7 cm2/s, Mw = 92 kD. (B) EriC: s = 8.43 S, D = 6.47 × 10−7 cm2/s, Mw = 126 kD.
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Figure 8: Velocity sedimentation of Eric. A280 in each sample cell is plotted as a function of radius from the axis of rotation. Each scan was made at a different time during the centrifugation run; the scans made at 27, 107, and 187 min are specifically labeled. Fits to the Fujita-MacCosham function yields values for the sedimentation coefficient, s, and diffusion coefficient, D, which in turn yield estimates of molar mass, Mw. (A) KcsA: s = 6.73 S, D = 6.55 × 10−7 cm2/s, Mw = 92 kD. (B) EriC: s = 8.43 S, D = 6.47 × 10−7 cm2/s, Mw = 126 kD.

Mentions: Since these gel filtration results cannot distinguish dimers from higher-order oligomers, we also analyzed velocity sedimentation profiles of EriC and again used KcsA as a membrane protein size standard. Qualitatively, EriC sediments more rapidly than KcsA (Fig. 8). A fit of the sedimentation data to a Fujita function (Williams 1972), which determines both the sedimentation and diffusion coefficients and hence molecular mass, yields 92 kD for KcsA and 126 kD for EriC. These estimates are both ∼25% higher than the formula size of tetrameric KcsA and dimeric EriC, a result easily rationalized by bound detergent. A value of 126 kD for EriC would be difficult to reconcile with a trimer or higher oligomer.


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)

Velocity sedimentation of Eric. A280 in each sample cell is plotted as a function of radius from the axis of rotation. Each scan was made at a different time during the centrifugation run; the scans made at 27, 107, and 187 min are specifically labeled. Fits to the Fujita-MacCosham function yields values for the sedimentation coefficient, s, and diffusion coefficient, D, which in turn yield estimates of molar mass, Mw. (A) KcsA: s = 6.73 S, D = 6.55 × 10−7 cm2/s, Mw = 92 kD. (B) EriC: s = 8.43 S, D = 6.47 × 10−7 cm2/s, Mw = 126 kD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230540&req=5

Figure 8: Velocity sedimentation of Eric. A280 in each sample cell is plotted as a function of radius from the axis of rotation. Each scan was made at a different time during the centrifugation run; the scans made at 27, 107, and 187 min are specifically labeled. Fits to the Fujita-MacCosham function yields values for the sedimentation coefficient, s, and diffusion coefficient, D, which in turn yield estimates of molar mass, Mw. (A) KcsA: s = 6.73 S, D = 6.55 × 10−7 cm2/s, Mw = 92 kD. (B) EriC: s = 8.43 S, D = 6.47 × 10−7 cm2/s, Mw = 126 kD.
Mentions: Since these gel filtration results cannot distinguish dimers from higher-order oligomers, we also analyzed velocity sedimentation profiles of EriC and again used KcsA as a membrane protein size standard. Qualitatively, EriC sediments more rapidly than KcsA (Fig. 8). A fit of the sedimentation data to a Fujita function (Williams 1972), which determines both the sedimentation and diffusion coefficients and hence molecular mass, yields 92 kD for KcsA and 126 kD for EriC. These estimates are both ∼25% higher than the formula size of tetrameric KcsA and dimeric EriC, a result easily rationalized by bound detergent. A value of 126 kD for EriC would be difficult to reconcile with a trimer or higher oligomer.

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