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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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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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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.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