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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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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.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