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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

Quantification of EriC activity. The fraction of vesicles containing no active Cl− channels, f0, was determined for vesicles (3.8 mg lipid) reconstituted with the indicated amount of EriC. The data were fit to  as described in the text. Parameters of the fit were: ME = 102,000 g/mol; θ = 5.2 cm3/g (±0.2, n = 26); σ = 2.3 × 106 cm2/g. This latter parameter was calculated from molecular surface area, 61 Å2 (Rand and Parsegian 1989), an estimated molecular weight of the phospholipid (750 g/mol), and a factor of 0.93 to account for liposome curvature.
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Figure 4: Quantification of EriC activity. The fraction of vesicles containing no active Cl− channels, f0, was determined for vesicles (3.8 mg lipid) reconstituted with the indicated amount of EriC. The data were fit to as described in the text. Parameters of the fit were: ME = 102,000 g/mol; θ = 5.2 cm3/g (±0.2, n = 26); σ = 2.3 × 106 cm2/g. This latter parameter was calculated from molecular surface area, 61 Å2 (Rand and Parsegian 1989), an estimated molecular weight of the phospholipid (750 g/mol), and a factor of 0.93 to account for liposome curvature.

Mentions: As EriC is reconstituted at increasing protein/lipid mass ratio, the fraction of Cl−-impermeable volume, f0, falls exponentially from a protein-free value of unity (Fig. 4), as demanded by a Poisson distribution governing the random incorporation of NE EriC channels into NL liposomes (Goldberg and Miller 1991). The absolute value of intravesicular volume, θ, does not change systematically over this range of protein concentration (data not shown), which roughly spans the range of 0.2–4 channels per liposome. The protein concentration dependence of f0 quantitatively obeys the expectations of , where the crucial fit quantity, the fraction of active protein, is s = 1.9. Since we have based our analysis on a utopian model in which protein molecules distribute perfectly into uniform spherical vesicles, it is perhaps not surprising that we reach a physically impossible conclusion of 190% activity. As discussed below, several assumptions in this analysis are expected to lead to uncertainty in the estimation of EriC activity, so we can easily rationalize this absurd value. The important point of this calculation is that a major fraction of the purified EriC protein—perhaps 100%—is responsible for the functional activity detected in the assay.


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)

Quantification of EriC activity. The fraction of vesicles containing no active Cl− channels, f0, was determined for vesicles (3.8 mg lipid) reconstituted with the indicated amount of EriC. The data were fit to  as described in the text. Parameters of the fit were: ME = 102,000 g/mol; θ = 5.2 cm3/g (±0.2, n = 26); σ = 2.3 × 106 cm2/g. This latter parameter was calculated from molecular surface area, 61 Å2 (Rand and Parsegian 1989), an estimated molecular weight of the phospholipid (750 g/mol), and a factor of 0.93 to account for liposome curvature.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Quantification of EriC activity. The fraction of vesicles containing no active Cl− channels, f0, was determined for vesicles (3.8 mg lipid) reconstituted with the indicated amount of EriC. The data were fit to as described in the text. Parameters of the fit were: ME = 102,000 g/mol; θ = 5.2 cm3/g (±0.2, n = 26); σ = 2.3 × 106 cm2/g. This latter parameter was calculated from molecular surface area, 61 Å2 (Rand and Parsegian 1989), an estimated molecular weight of the phospholipid (750 g/mol), and a factor of 0.93 to account for liposome curvature.
Mentions: As EriC is reconstituted at increasing protein/lipid mass ratio, the fraction of Cl−-impermeable volume, f0, falls exponentially from a protein-free value of unity (Fig. 4), as demanded by a Poisson distribution governing the random incorporation of NE EriC channels into NL liposomes (Goldberg and Miller 1991). The absolute value of intravesicular volume, θ, does not change systematically over this range of protein concentration (data not shown), which roughly spans the range of 0.2–4 channels per liposome. The protein concentration dependence of f0 quantitatively obeys the expectations of , where the crucial fit quantity, the fraction of active protein, is s = 1.9. Since we have based our analysis on a utopian model in which protein molecules distribute perfectly into uniform spherical vesicles, it is perhaps not surprising that we reach a physically impossible conclusion of 190% activity. As discussed below, several assumptions in this analysis are expected to lead to uncertainty in the estimation of EriC activity, so we can easily rationalize this absurd value. The important point of this calculation is that a major fraction of the purified EriC protein—perhaps 100%—is responsible for the functional activity detected in the assay.

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