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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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Ion selectivity of EriC. (A) Effect of internal ion on uptake of 36Cl−. Vesicles were reconstituted with 1.9 μg EriC/mg lipid in 450 mM KX, 20 mM Na-MES, pH 6.2. Uptake was measured at 13 min; data points represent mean ± SEM of three or range of two measurements. (B) Effect of external ions on 36Cl− uptake. Vesicles were reconstituted with 3.7 (10 mM test ion) or 3.0 (19 and 50 mM test ion) μg EriC/mg lipid. Uptake was measured at 13 min. Data (mean ± SEM, n = 3), are normalized to uptake with no test ion added. glu−, glutamate.
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Figure 5: Ion selectivity of EriC. (A) Effect of internal ion on uptake of 36Cl−. Vesicles were reconstituted with 1.9 μg EriC/mg lipid in 450 mM KX, 20 mM Na-MES, pH 6.2. Uptake was measured at 13 min; data points represent mean ± SEM of three or range of two measurements. (B) Effect of external ions on 36Cl− uptake. Vesicles were reconstituted with 3.7 (10 mM test ion) or 3.0 (19 and 50 mM test ion) μg EriC/mg lipid. Uptake was measured at 13 min. Data (mean ± SEM, n = 3), are normalized to uptake with no test ion added. glu−, glutamate.

Mentions: The concentrative uptake assay was used in two ways to gauge the ionic selectivity of EriC. In the first set of experiments, internal Cl− was replaced with various test anions. Permeant anions support concentrative uptake, while impermeant anions do not. Of the anions tested (Fig. 5 A), only Cl−, Br−, and NO3− are permeant by this criterion. In the second set of experiments, we added test ions to the external solution to see which of these would collapse the liposome membrane potential and thereby impede influx. In this assay, SCN−, and to a lesser extent I− and F− score as permeant in addition to Cl−, Br−, and NO3−. The discrepancy observed with SCN− is not disconcerting; SCN− both blocks and permeates some eukaryotic ClCs (White and Miller 1981; Fahlke et al. 1997b; Rychov et al. 1998) and, if acting in such a fashion here, would also inhibit influx. In other words, the flux assay in which the test anion is applied externally does not distinguish a permeant ion from a strong blocker. In any case, the two assays taken together demonstrate (a) that EriC-mediated fluxes are highly selective for anions over cations, and (b) that among anions the selectivity sequence is roughly similar to that found for ClC-0 and ClC-1 (Rychov et al. 1998; Jentsch et al. 1999): Cl−, Br− > NO3− > I−, F− >> H2PO4−, glutamate−. It is worth noting, however, that these selectivity assays are performed under ionic conditions and with methods very different than in electrophysiological measurements, and that no single interanionic selectivity sequence applies to all eukaryotic ClC channels.


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)

Ion selectivity of EriC. (A) Effect of internal ion on uptake of 36Cl−. Vesicles were reconstituted with 1.9 μg EriC/mg lipid in 450 mM KX, 20 mM Na-MES, pH 6.2. Uptake was measured at 13 min; data points represent mean ± SEM of three or range of two measurements. (B) Effect of external ions on 36Cl− uptake. Vesicles were reconstituted with 3.7 (10 mM test ion) or 3.0 (19 and 50 mM test ion) μg EriC/mg lipid. Uptake was measured at 13 min. Data (mean ± SEM, n = 3), are normalized to uptake with no test ion added. glu−, glutamate.
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Related In: Results  -  Collection

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Figure 5: Ion selectivity of EriC. (A) Effect of internal ion on uptake of 36Cl−. Vesicles were reconstituted with 1.9 μg EriC/mg lipid in 450 mM KX, 20 mM Na-MES, pH 6.2. Uptake was measured at 13 min; data points represent mean ± SEM of three or range of two measurements. (B) Effect of external ions on 36Cl− uptake. Vesicles were reconstituted with 3.7 (10 mM test ion) or 3.0 (19 and 50 mM test ion) μg EriC/mg lipid. Uptake was measured at 13 min. Data (mean ± SEM, n = 3), are normalized to uptake with no test ion added. glu−, glutamate.
Mentions: The concentrative uptake assay was used in two ways to gauge the ionic selectivity of EriC. In the first set of experiments, internal Cl− was replaced with various test anions. Permeant anions support concentrative uptake, while impermeant anions do not. Of the anions tested (Fig. 5 A), only Cl−, Br−, and NO3− are permeant by this criterion. In the second set of experiments, we added test ions to the external solution to see which of these would collapse the liposome membrane potential and thereby impede influx. In this assay, SCN−, and to a lesser extent I− and F− score as permeant in addition to Cl−, Br−, and NO3−. The discrepancy observed with SCN− is not disconcerting; SCN− both blocks and permeates some eukaryotic ClCs (White and Miller 1981; Fahlke et al. 1997b; Rychov et al. 1998) and, if acting in such a fashion here, would also inhibit influx. In other words, the flux assay in which the test anion is applied externally does not distinguish a permeant ion from a strong blocker. In any case, the two assays taken together demonstrate (a) that EriC-mediated fluxes are highly selective for anions over cations, and (b) that among anions the selectivity sequence is roughly similar to that found for ClC-0 and ClC-1 (Rychov et al. 1998; Jentsch et al. 1999): Cl−, Br− > NO3− > I−, F− >> H2PO4−, glutamate−. It is worth noting, however, that these selectivity assays are performed under ionic conditions and with methods very different than in electrophysiological measurements, and that no single interanionic selectivity sequence applies to all eukaryotic ClC channels.

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