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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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Conservation of prokaryotic ClC channels. Regions of protein that contain residues invariant among mammalian or all eukaryotic ClC channels are color-coded and mapped onto the ClC transmembrane topology model of Schmidt-Rose and Jentsch 1997. The topology of transmembrane stretches 5–7 is currently unresolved (Fahlke et al. 1997c; Jentsch et al. 1999). The COOH-terminal cytoplasmic domain, which contains no invariant regions, ranges from ∼170 to 420 residues in the mammalian ClCs and 6 to 475 residues in the prokaryotes (35 residues in EriC). Corresponding sequences of the prokaryotic ClC genes identified by BLAST searches are shown below the eukaryotic and mammalian consensus sequences (x, variant residue). Prokaryotic species and sequence references are as follows. Ec, Escherichia coli (Blattner et al. 1997); Syn, Synechocystis sp. strain PCC 6803 (Kaneko et al. 1996); Vc, Vibrio cholerae (TIGR, preliminary sequence data); Ph, Pyrococcus horikoshii (Kawarabayasi et al. 1998); Mt, Mycobacterium tuberculosis (Cole et al. 1998); Af, Archaeoglobus fulgidus (Klenk et al. 1997); Mj, Methanococcus jannaschii (Bult et al. 1996). EriC corresponds to Ec 1.
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Figure 1: Conservation of prokaryotic ClC channels. Regions of protein that contain residues invariant among mammalian or all eukaryotic ClC channels are color-coded and mapped onto the ClC transmembrane topology model of Schmidt-Rose and Jentsch 1997. The topology of transmembrane stretches 5–7 is currently unresolved (Fahlke et al. 1997c; Jentsch et al. 1999). The COOH-terminal cytoplasmic domain, which contains no invariant regions, ranges from ∼170 to 420 residues in the mammalian ClCs and 6 to 475 residues in the prokaryotes (35 residues in EriC). Corresponding sequences of the prokaryotic ClC genes identified by BLAST searches are shown below the eukaryotic and mammalian consensus sequences (x, variant residue). Prokaryotic species and sequence references are as follows. Ec, Escherichia coli (Blattner et al. 1997); Syn, Synechocystis sp. strain PCC 6803 (Kaneko et al. 1996); Vc, Vibrio cholerae (TIGR, preliminary sequence data); Ph, Pyrococcus horikoshii (Kawarabayasi et al. 1998); Mt, Mycobacterium tuberculosis (Cole et al. 1998); Af, Archaeoglobus fulgidus (Klenk et al. 1997); Mj, Methanococcus jannaschii (Bult et al. 1996). EriC corresponds to Ec 1.

Mentions: For the past few years, it has been appreciated that ClC channels are represented in microbial genomes (Jentsch et al. 1999). By sequence identity, the prokaryotic ClCs lie in the twilight zone for homology modeling (∼15–25%), but the statistical scores obtained from BLAST alignments indicate a high probability that the prokaryotic proteins are true ClC homologues (Abagyan and Batalov 1997; Brenner et al. 1998). Manual scrutiny of the prokaryotic sequences supports this conclusion. Several stretches of ClC sequence are invariant among the known eukaryotic family members, and these appear throughout the entire membrane-spanning region, ∼400 residues, and only in this region. In these regions, the prokaryotic proteins display high sequence identity to their eukaryotic counterparts (Fig. 1). The roles that these conserved sequences play in channel function have not been experimentally established; however, certain of these, when mutated, lead to changes in both gating and permeation (Fahlke et al. 1997b,Fahlke et al. 1997d; Ludewig et al. 1997), as though they either directly mediate these functions or are necessary for proper folding and assembly of 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)

Conservation of prokaryotic ClC channels. Regions of protein that contain residues invariant among mammalian or all eukaryotic ClC channels are color-coded and mapped onto the ClC transmembrane topology model of Schmidt-Rose and Jentsch 1997. The topology of transmembrane stretches 5–7 is currently unresolved (Fahlke et al. 1997c; Jentsch et al. 1999). The COOH-terminal cytoplasmic domain, which contains no invariant regions, ranges from ∼170 to 420 residues in the mammalian ClCs and 6 to 475 residues in the prokaryotes (35 residues in EriC). Corresponding sequences of the prokaryotic ClC genes identified by BLAST searches are shown below the eukaryotic and mammalian consensus sequences (x, variant residue). Prokaryotic species and sequence references are as follows. Ec, Escherichia coli (Blattner et al. 1997); Syn, Synechocystis sp. strain PCC 6803 (Kaneko et al. 1996); Vc, Vibrio cholerae (TIGR, preliminary sequence data); Ph, Pyrococcus horikoshii (Kawarabayasi et al. 1998); Mt, Mycobacterium tuberculosis (Cole et al. 1998); Af, Archaeoglobus fulgidus (Klenk et al. 1997); Mj, Methanococcus jannaschii (Bult et al. 1996). EriC corresponds to Ec 1.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Conservation of prokaryotic ClC channels. Regions of protein that contain residues invariant among mammalian or all eukaryotic ClC channels are color-coded and mapped onto the ClC transmembrane topology model of Schmidt-Rose and Jentsch 1997. The topology of transmembrane stretches 5–7 is currently unresolved (Fahlke et al. 1997c; Jentsch et al. 1999). The COOH-terminal cytoplasmic domain, which contains no invariant regions, ranges from ∼170 to 420 residues in the mammalian ClCs and 6 to 475 residues in the prokaryotes (35 residues in EriC). Corresponding sequences of the prokaryotic ClC genes identified by BLAST searches are shown below the eukaryotic and mammalian consensus sequences (x, variant residue). Prokaryotic species and sequence references are as follows. Ec, Escherichia coli (Blattner et al. 1997); Syn, Synechocystis sp. strain PCC 6803 (Kaneko et al. 1996); Vc, Vibrio cholerae (TIGR, preliminary sequence data); Ph, Pyrococcus horikoshii (Kawarabayasi et al. 1998); Mt, Mycobacterium tuberculosis (Cole et al. 1998); Af, Archaeoglobus fulgidus (Klenk et al. 1997); Mj, Methanococcus jannaschii (Bult et al. 1996). EriC corresponds to Ec 1.
Mentions: For the past few years, it has been appreciated that ClC channels are represented in microbial genomes (Jentsch et al. 1999). By sequence identity, the prokaryotic ClCs lie in the twilight zone for homology modeling (∼15–25%), but the statistical scores obtained from BLAST alignments indicate a high probability that the prokaryotic proteins are true ClC homologues (Abagyan and Batalov 1997; Brenner et al. 1998). Manual scrutiny of the prokaryotic sequences supports this conclusion. Several stretches of ClC sequence are invariant among the known eukaryotic family members, and these appear throughout the entire membrane-spanning region, ∼400 residues, and only in this region. In these regions, the prokaryotic proteins display high sequence identity to their eukaryotic counterparts (Fig. 1). The roles that these conserved sequences play in channel function have not been experimentally established; however, certain of these, when mutated, lead to changes in both gating and permeation (Fahlke et al. 1997b,Fahlke et al. 1997d; Ludewig et al. 1997), as though they either directly mediate these functions or are necessary for proper folding and assembly of 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