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Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule.

Engh AM, Maduke M - J. Gen. Physiol. (2005)

Bottom Line: Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels.When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004).These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305, USA.

ABSTRACT
ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.

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Candidate pore-lining secondary structures. (A) Structure of ClC-ec1 (1OTS) as viewed from within the membrane. The subunits are colored gray and cyan, with secondary structures lining the intracellular vestibule of the gray subunit colored as follows: helix C and loop CD, orange; helix D and loop DE, magenta; helix E, light pink; helix F, periwinkle; helix J, royal blue; helix M, tan; helix R, red. The figure was created using Pymol (DeLano, 2002). (B) Alignment of secondary structures screened. ClC-ec1 and ClC-0 sequences were aligned using Clustal-W, with slight manual adjustment. Conserved residues are shaded. The regions screened are indicated by bars above the ClC-0 sequence. The beginning of helix J is omitted.
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fig1: Candidate pore-lining secondary structures. (A) Structure of ClC-ec1 (1OTS) as viewed from within the membrane. The subunits are colored gray and cyan, with secondary structures lining the intracellular vestibule of the gray subunit colored as follows: helix C and loop CD, orange; helix D and loop DE, magenta; helix E, light pink; helix F, periwinkle; helix J, royal blue; helix M, tan; helix R, red. The figure was created using Pymol (DeLano, 2002). (B) Alignment of secondary structures screened. ClC-ec1 and ClC-0 sequences were aligned using Clustal-W, with slight manual adjustment. Conserved residues are shaded. The regions screened are indicated by bars above the ClC-0 sequence. The beginning of helix J is omitted.

Mentions: A cytoplasmic view of the E. coli ClC structure suggests at least six helices and two loops that might line the inner vestibule of the ClC-0 channel (Fig. 1 A, helices C, D, F, J, M, R, loop CD, DE). In addition, recent in silico studies of the prokaryotic ClC structures suggest specific residues on these sequence elements that are pore lining, and indicate that helix E may play a role in permeation (Corry et al., 2004; Faraldo-Gomez and Roux, 2004; Miloshevsky and Jordan, 2004). Previous studies have identified S123, E127, and residues along helix R as likely to be pore lining in ClC-0 (Pusch et al., 1995; Ludewig et al., 1996, 1997c; Middleton et al., 1996; Lin and Chen, 2000, 2003; Chen and Chen, 2003; Chen et al., 2003). To flesh out the picture of the intracellular vestibule, we screened three sequence elements in ClC-0 as well as nine residues on four other sequence elements hypothesized to line the intracellular vestibule (Fig. 1, A and B).


Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule.

Engh AM, Maduke M - J. Gen. Physiol. (2005)

Candidate pore-lining secondary structures. (A) Structure of ClC-ec1 (1OTS) as viewed from within the membrane. The subunits are colored gray and cyan, with secondary structures lining the intracellular vestibule of the gray subunit colored as follows: helix C and loop CD, orange; helix D and loop DE, magenta; helix E, light pink; helix F, periwinkle; helix J, royal blue; helix M, tan; helix R, red. The figure was created using Pymol (DeLano, 2002). (B) Alignment of secondary structures screened. ClC-ec1 and ClC-0 sequences were aligned using Clustal-W, with slight manual adjustment. Conserved residues are shaded. The regions screened are indicated by bars above the ClC-0 sequence. The beginning of helix J is omitted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234078&req=5

fig1: Candidate pore-lining secondary structures. (A) Structure of ClC-ec1 (1OTS) as viewed from within the membrane. The subunits are colored gray and cyan, with secondary structures lining the intracellular vestibule of the gray subunit colored as follows: helix C and loop CD, orange; helix D and loop DE, magenta; helix E, light pink; helix F, periwinkle; helix J, royal blue; helix M, tan; helix R, red. The figure was created using Pymol (DeLano, 2002). (B) Alignment of secondary structures screened. ClC-ec1 and ClC-0 sequences were aligned using Clustal-W, with slight manual adjustment. Conserved residues are shaded. The regions screened are indicated by bars above the ClC-0 sequence. The beginning of helix J is omitted.
Mentions: A cytoplasmic view of the E. coli ClC structure suggests at least six helices and two loops that might line the inner vestibule of the ClC-0 channel (Fig. 1 A, helices C, D, F, J, M, R, loop CD, DE). In addition, recent in silico studies of the prokaryotic ClC structures suggest specific residues on these sequence elements that are pore lining, and indicate that helix E may play a role in permeation (Corry et al., 2004; Faraldo-Gomez and Roux, 2004; Miloshevsky and Jordan, 2004). Previous studies have identified S123, E127, and residues along helix R as likely to be pore lining in ClC-0 (Pusch et al., 1995; Ludewig et al., 1996, 1997c; Middleton et al., 1996; Lin and Chen, 2000, 2003; Chen and Chen, 2003; Chen et al., 2003). To flesh out the picture of the intracellular vestibule, we screened three sequence elements in ClC-0 as well as nine residues on four other sequence elements hypothesized to line the intracellular vestibule (Fig. 1, A and B).

Bottom Line: Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels.When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004).These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305, USA.

ABSTRACT
ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.

Show MeSH