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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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36Cl− flux through reconstituted EriC. Concentrative uptake of 36Cl− was followed as in materials and methods. (A) Time course of accumulation of 36Cl− in vesicles reconstituted with 4.5 μg EriC/mg lipid (•, mean ± SEM, n = 4) or without protein (▪, n = 1). 36Cl− release was measured after addition of valinomycin at 21 min (○, n = 1). (B) Protein concentration-dependent accumulation of 36Cl− into vesicles reconstituted with EriC (○, •) or KcsA (▪). Uptake was measured at 20 min (mean ± SEM, n = 3).
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Figure 3: 36Cl− flux through reconstituted EriC. Concentrative uptake of 36Cl− was followed as in materials and methods. (A) Time course of accumulation of 36Cl− in vesicles reconstituted with 4.5 μg EriC/mg lipid (•, mean ± SEM, n = 4) or without protein (▪, n = 1). 36Cl− release was measured after addition of valinomycin at 21 min (○, n = 1). (B) Protein concentration-dependent accumulation of 36Cl− into vesicles reconstituted with EriC (○, •) or KcsA (▪). Uptake was measured at 20 min (mean ± SEM, n = 3).

Mentions: EriC is unambiguously a ClC-family protein at the level of primary sequence, but does it function as an ion channel? To assess this protein's ability to catalyze passive transmembrane flux of 36Cl−, we reconstituted EriC into liposomes and performed a concentrative influx assay (Goldberg and Miller 1991). Vesicles were loaded with a high concentration (450 mM) of Cl−, and 36Cl− influx was measured at low (2.5 mM) external Cl−. The resulting Cl− gradient will polarize any Cl−-selective liposome membrane (positive potential inside), and 36Cl− will accordingly accumulate inside the liposome. Liposomes that have failed to incorporate Cl− channels or have sprung nonselective leaks will not accumulate 36Cl− in this assay. The results of Fig. 3 demonstrate that EriC is a Cl−-selective channel; tracer Cl− accumulates into EriC-containing vesicles with a time constant of ∼4 min. Permeabilization of the liposomes to K+ with valinomycin causes rapid loss of Cl−, a result demonstrating that the movement of 36Cl− is conductive, not electroneutral. The final level of tracer rises in a saturating fashion with the amount of EriC reconstituted, as the population of channel-containing liposomes increases. Control protein-free vesicles or vesicles reconstituted with KcsA, a bacterial K+ channel, fail to transport 36Cl−, although the latter vesicles accumulate 86Rb+ under similar conditions (Heginbotham et al. 1998).


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)

36Cl− flux through reconstituted EriC. Concentrative uptake of 36Cl− was followed as in materials and methods. (A) Time course of accumulation of 36Cl− in vesicles reconstituted with 4.5 μg EriC/mg lipid (•, mean ± SEM, n = 4) or without protein (▪, n = 1). 36Cl− release was measured after addition of valinomycin at 21 min (○, n = 1). (B) Protein concentration-dependent accumulation of 36Cl− into vesicles reconstituted with EriC (○, •) or KcsA (▪). Uptake was measured at 20 min (mean ± SEM, n = 3).
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Related In: Results  -  Collection

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Figure 3: 36Cl− flux through reconstituted EriC. Concentrative uptake of 36Cl− was followed as in materials and methods. (A) Time course of accumulation of 36Cl− in vesicles reconstituted with 4.5 μg EriC/mg lipid (•, mean ± SEM, n = 4) or without protein (▪, n = 1). 36Cl− release was measured after addition of valinomycin at 21 min (○, n = 1). (B) Protein concentration-dependent accumulation of 36Cl− into vesicles reconstituted with EriC (○, •) or KcsA (▪). Uptake was measured at 20 min (mean ± SEM, n = 3).
Mentions: EriC is unambiguously a ClC-family protein at the level of primary sequence, but does it function as an ion channel? To assess this protein's ability to catalyze passive transmembrane flux of 36Cl−, we reconstituted EriC into liposomes and performed a concentrative influx assay (Goldberg and Miller 1991). Vesicles were loaded with a high concentration (450 mM) of Cl−, and 36Cl− influx was measured at low (2.5 mM) external Cl−. The resulting Cl− gradient will polarize any Cl−-selective liposome membrane (positive potential inside), and 36Cl− will accordingly accumulate inside the liposome. Liposomes that have failed to incorporate Cl− channels or have sprung nonselective leaks will not accumulate 36Cl− in this assay. The results of Fig. 3 demonstrate that EriC is a Cl−-selective channel; tracer Cl− accumulates into EriC-containing vesicles with a time constant of ∼4 min. Permeabilization of the liposomes to K+ with valinomycin causes rapid loss of Cl−, a result demonstrating that the movement of 36Cl− is conductive, not electroneutral. The final level of tracer rises in a saturating fashion with the amount of EriC reconstituted, as the population of channel-containing liposomes increases. Control protein-free vesicles or vesicles reconstituted with KcsA, a bacterial K+ channel, fail to transport 36Cl−, although the latter vesicles accumulate 86Rb+ under similar conditions (Heginbotham et al. 1998).

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