Escherichia coli K-12 substr. MG1655 Transporter: chloride:H+ antiporter ClcA

Gene: clcA Accession Numbers: EG12331 (EcoCyc), b0155, ECK0154

Synonyms: eriC, yadQ, ClC-ec1

Regulation Summary Diagram: ?

Regulation summary diagram for clcA

Subunit composition of chloride:H+ antiporter ClcA = [ClcA]2

ClcA is a member of the chloride carrier/channel (ClC) family and mediates a chloride:proton antiport reaction (a secondary carrier type transport reaction) in which two Cl- exchange for one H+. ClcA forms part of E. coli's response to extreme acid conditions; ClcA promotes H+ extrusion in response to the persistant proton influx that occurs in a strongly acidic environment [Iyer02, Accardi04, Miller09].

ClcA exists as a homodimer, each monomer contains an ion conducting pore and selectivity filter. The ClcA monomer contains 18 α-helices and exhibits an antiparallel orientation, that is, the two halves of the monomer have opposite orientations in the membrane [Dutzler02]. Analysis of the crystal structure at resolution of 2.5 angstroms of ClcA bound to a Fab fragment revealed that gating occurs independently within each monomer and is pH-dependent [Dutzler03]. Cl-/H+ exchange is preserved in ClcA dimers constrained by covalent cross-links across their dimer interface [Nguitragool07] and in ClcA mutants in which the dimer interface is destabilised [Robertson10] suggesting that the ClcA monomer contains all the essential components of the transport mechanism.

3 critical glutamate residues have been identified. E203 (known as Gluinternal) and E148 (Gluexternal) are believed to act as waystations for H+ as it travels through the protein. Mutation of either to non-protonable residues abolishes H+ transport while preserving Cl- transport [Accardi04, Accardi05]. E202 mutation by large aromatic substitution results in severe slowing of the antiport rate due to disruption of H+ diffusion between the intracellular solution and Gluinternal [Lim12].

A detailed topology map for ClcA has been reported [Cassel08].

Cells lacking both ClcA and ClcB do not survive extreme acid shock conditions (pH2.5, 2 hrs at 37°C in minimal medium); expression of clcA from a low-copy number plasmid rescues this phenotype [Iyer02].

Reviews: [Matulef, Accardi10]

Citations: [Miloshevsky10, Saier99, Jayaram08, Lim09]

Locations: inner membrane

Map Position: [175,107 -> 176,528] (3.77 centisomes, 14°)
Length: 1422 bp / 473 aa

Molecular Weight of Polypeptide: 50.349 kD (from nucleotide sequence), 38.0 kD (experimental) [Maduke99 ]

Unification Links: ASAP:ABE-0000533 , DIP:DIP-9523N , EchoBASE:EB2235 , EcoGene:EG12331 , EcoliWiki:b0155 , ModBase:P37019 , OU-Microarray:b0155 , PDB:1KPK , PDB:1OTS , PDB:1OTT , PDB:1OTU , PDB:2EXW , PDB:2EXY , PDB:2EZ0 , PDB:2FEC , PDB:2FED , PDB:2FEE , PDB:2H2P , PDB:2H2S , PDB:2HLF , PDB:2HT2 , PDB:2HT3 , PDB:2HT4 , PDB:2HTK , PDB:2HTL , PDB:2R9H , PDB:3DET , PDB:3EJY , PDB:3EJZ , PDB:3NMO , PortEco:clcA , PR:PRO_000022294 , Protein Model Portal:P37019 , RefSeq:NP_414697 , RegulonDB:EG12331 , SMR:P37019 , String:511145.b0155 , UniProt:P37019

Relationship Links: InterPro:IN-FAMILY:IPR001807 , InterPro:IN-FAMILY:IPR014743 , InterPro:IN-FAMILY:IPR023861 , PDB:Structure:4ENE , PDB:Structure:4FTP , PDB:Structure:4KJP , PDB:Structure:4KJQ , PDB:Structure:4KJW , PDB:Structure:4KK5 , PDB:Structure:4KK6 , PDB:Structure:4KK8 , PDB:Structure:4KK9 , PDB:Structure:4KKA , PDB:Structure:4KKB , PDB:Structure:4KKC , PDB:Structure:4KKL , PDB:Structure:4LOU , PDB:Structure:4MQX , Pfam:IN-FAMILY:PF00654 , Prints:IN-FAMILY:PR00762

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0006821 - chloride transport Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, Maduke99, Accardi04]
GO:0015706 - nitrate transport Inferred from experiment [Nguitragool06]
GO:0071468 - cellular response to acidic pH Inferred from experiment [Iyer02]
GO:1902600 - hydrogen ion transmembrane transport Inferred from experiment [Accardi04]
GO:0006810 - transport Inferred by computational analysis [UniProtGOA11a]
GO:0006811 - ion transport Inferred by computational analysis [UniProtGOA11a]
GO:0034220 - ion transmembrane transport Inferred by computational analysis [GOA01a]
GO:0055085 - transmembrane transport Inferred by computational analysis [GOA01a]
GO:1902476 - chloride transmembrane transport Inferred by computational analysis [GOA01a]
GO:1903959 - regulation of anion transmembrane transport Inferred by computational analysis [GOA01a]
Molecular Function: GO:0015299 - solute:proton antiporter activity Inferred from experiment [Accardi04]
GO:0031404 - chloride ion binding Inferred from experiment [Maduke99, Dutzler03]
GO:0043168 - anion binding Inferred from experiment [Nguitragool06, Lim09]
GO:0005216 - ion channel activity Inferred by computational analysis [GOA01a]
GO:0005247 - voltage-gated chloride channel activity Inferred by computational analysis [GOA01a]
GO:0015108 - chloride transmembrane transporter activity Inferred by computational analysis [GOA01a]
GO:0015297 - antiporter activity Inferred by computational analysis [UniProtGOA11a, GOA06]
Cellular Component: GO:0005886 - plasma membrane Inferred from experiment Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06, DiazMejia09, Zhang07, Daley05]
GO:0005887 - integral component of plasma membrane Inferred from experiment Inferred by computational analysis [GOA01a, Maduke99]
GO:0016020 - membrane Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016021 - integral component of membrane Inferred by computational analysis [UniProtGOA11a]

MultiFun Terms: transport Channel-type Transporters alpha-type channels

Essentiality data for clcA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Created 21-Dec-2011 by Mackie A , Macquarie University
Last-Curated ? 31-May-2015 by Mackie A , Macquarie University

Enzymatic reaction of: chloride:H+ antiporter ClcA

Transport reaction diagram for chloride:H+ antiporter ClcA

Alternative Products for chloride: nitrate [Nguitragool06 ] , bromide [Nguitragool06 ]

Sequence Features

Protein sequence of ClcA with features indicated

Feature Class Location Citations Comment
Sequence-Conflict 32
[Fujita94, UniProt10]
UniProt: (in Ref. 1);
Transmembrane-Region 33 -> 69
UniProt: Helical.
Transmembrane-Region 77 -> 100
UniProt: Helical.
Protein-Segment 106 -> 110
UniProt: Selectivity filter part_1; Sequence Annotation Type: short sequence motif.
Amino-Acid-Sites-That-Bind 107
UniProt: Chloride.
Mutagenesis-Variant 107
[Lobet06, UniProt11]
UniProt: Uncouples chloride transport from proton transport.
Intramembrane-Region 109 -> 116
UniProt: Helical.
Transmembrane-Region 124 -> 141
UniProt: Helical.
Protein-Segment 146 -> 150
UniProt: Selectivity filter part_2; Sequence Annotation Type: short sequence motif.
Amino-Acid-Site 148
UniProt: Mediates proton transfer from the outer aqueous phase to the interior of the protein; involved in linking H(+) and Cl(-) transport; Sequence Annotation Type: site.
Transmembrane-Region 148 -> 166
UniProt: Helical.
Mutagenesis-Variant 148
[Jayaram08, Lobet06, Dutzler03, Accardi04, UniProt11]
E → A or Q: Abolishes proton transport, but permits the transit of chloride ions. Abolishes gating, permitting continuous rapid transit of chloride ions; when associated with A-445.
Intramembrane-Region 177 -> 189
UniProt: Helical.
Intramembrane-Region 193 -> 201
UniProt: Helical.
Amino-Acid-Site 203
UniProt: Mediates proton transfer from the protein to the inner aqueous phase; Sequence Annotation Type: site.
Mutagenesis-Variant 203
[Lim09, UniProt11]
[UniProt11, Lim09, UniProt11]
[Lim09, UniProt11]
[Lim09, UniProt11]
E → C, I, L or V: Abolishes proton and chloride transport.
E → A, G, Q, S or T: Abolishes proton transport, and reduces chloride transport.
E → K or R: Decreased proton and chloride transport.
E → D or H: No effect on proton and chloride transport.
Transmembrane-Region 215 -> 232
UniProt: Helical.
Transmembrane-Region 253 -> 281
UniProt: Helical.
Transmembrane-Region 288 -> 309
UniProt: Helical.
Transmembrane-Region 330 -> 349
UniProt: Helical.
Protein-Segment 355 -> 359
UniProt: Selectivity filter part_3; Sequence Annotation Type: short sequence motif.
Transmembrane-Region 355 -> 376
UniProt: Helical.
Amino-Acid-Sites-That-Bind 356
UniProt: Chloride; via amide nitrogen.
Amino-Acid-Sites-That-Bind 357
UniProt: Chloride; via amide nitrogen.
Intramembrane-Region 387 -> 401
UniProt: Helical.
Intramembrane-Region 402 -> 404
UniProt: Note=Loop between two helices.
Intramembrane-Region 405 -> 416
UniProt: Helical.
Intramembrane-Region 417 -> 421
UniProt: Note=Loop between two helices.
Transmembrane-Region 422 -> 438
UniProt: Helical.
Amino-Acid-Sites-That-Bind 445
UniProt: Chloride.
Mutagenesis-Variant 445
[Jayaram08, Accardi06, Lobet06, UniProt11]
[Jayaram08, Accardi06, Lobet06, UniProt11]
[Jayaram08, Accardi06, Lobet06, UniProt11]
Y → A: Abolishes gating, permitting continuous rapid transit of chloride ions; when associated with A-148.
Y → L: Alters stoichiometry of proton/chloride exchange.
Y → F or W: No effect.

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b0155 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG12331; confirmed by SwissProt match.


Accardi04: Accardi A, Miller C (2004). "Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels." Nature 427(6977);803-7. PMID: 14985752

Accardi05: Accardi A, Walden M, Nguitragool W, Jayaram H, Williams C, Miller C (2005). "Separate ion pathways in a Cl-/H+ exchanger." J Gen Physiol 126(6);563-70. PMID: 16316975

Accardi06: Accardi A, Lobet S, Williams C, Miller C, Dutzler R (2006). "Synergism between halide binding and proton transport in a CLC-type exchanger." J Mol Biol 362(4);691-9. PMID: 16949616

Accardi10: Accardi A, Picollo A (2010). "CLC channels and transporters: proteins with borderline personalities." Biochim Biophys Acta 1798(8);1457-64. PMID: 20188062

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Cassel08: Cassel M, Seppala S, von Heijne G (2008). "Confronting fusion protein-based membrane protein topology mapping with reality: the Escherichia coli ClcA H+/Cl- exchange transporter." J Mol Biol 381(4);860-6. PMID: 18590742

Daley05: Daley DO, Rapp M, Granseth E, Melen K, Drew D, von Heijne G (2005). "Global topology analysis of the Escherichia coli inner membrane proteome." Science 308(5726);1321-3. PMID: 15919996

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Dutzler02: Dutzler R, Campbell EB, Cadene M, Chait BT, MacKinnon R (2002). "X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity." Nature 415(6869);287-94. PMID: 11796999

Dutzler03: Dutzler R, Campbell EB, MacKinnon R (2003). "Gating the selectivity filter in ClC chloride channels." Science 300(5616);108-12. PMID: 12649487

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Fujita94: Fujita N, Mori H, Yura T, Ishihama A (1994). "Systematic sequencing of the Escherichia coli genome: analysis of the 2.4-4.1 min (110,917-193,643 bp) region." Nucleic Acids Res 22(9);1637-9. PMID: 8202364

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Iyer02: Iyer R, Iverson TM, Accardi A, Miller C (2002). "A biological role for prokaryotic ClC chloride channels." Nature 419(6908);715-8. PMID: 12384697

Jayaram08: Jayaram H, Accardi A, Wu F, Williams C, Miller C (2008). "Ion permeation through a Cl--selective channel designed from a CLC Cl-/H+ exchanger." Proc Natl Acad Sci U S A 105(32);11194-9. PMID: 18678918

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Lim09: Lim HH, Miller C (2009). "Intracellular proton-transfer mutants in a CLC Cl-/H+ exchanger." J Gen Physiol 133(2);131-8. PMID: 19139174

Lim12: Lim HH, Shane T, Miller C (2012). "Intracellular proton access in a Cl(-)/H(+) antiporter." PLoS Biol 10(12);e1001441. PMID: 23239938

Lobet06: Lobet S, Dutzler R (2006). "Ion-binding properties of the ClC chloride selectivity filter." EMBO J 25(1);24-33. PMID: 16341087

Maduke99: Maduke M, Pheasant DJ, Miller C (1999). "High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel." J Gen Physiol 114(5);713-22. PMID: 10539975

Matulef: Matulef K, Maduke M "The CLC 'chloride channel' family: revelations from prokaryotes." Mol Membr Biol 24(5-6);342-50. PMID: 17710638

Miller09: Miller C, Nguitragool W (2009). "A provisional transport mechanism for a chloride channel-type Cl-/H+ exchanger." Philos Trans R Soc Lond B Biol Sci 364(1514);175-80. PMID: 18977737

Miloshevsky10: Miloshevsky GV, Hassanein A, Jordan PC (2010). "Antiport mechanism for Cl(-)/H(+) in ClC-ec1 from normal-mode analysis." Biophys J 98(6);999-1008. PMID: 20303857

Nguitragool06: Nguitragool W, Miller C (2006). "Uncoupling of a CLC Cl-/H+ exchange transporter by polyatomic anions." J Mol Biol 362(4);682-90. PMID: 16905147

Nguitragool07: Nguitragool W, Miller C (2007). "Inaugural Article: CLC Cl /H+ transporters constrained by covalent cross-linking." Proc Natl Acad Sci U S A 104(52);20659-65. PMID: 18093952

Robertson10: Robertson JL, Kolmakova-Partensky L, Miller C (2010). "Design, function and structure of a monomeric ClC transporter." Nature. PMID: 21048711

Saier99: Saier MH, Eng BH, Fard S, Garg J, Haggerty DA, Hutchinson WJ, Jack DL, Lai EC, Liu HJ, Nusinew DP, Omar AM, Pao SS, Paulsen IT, Quan JA, Sliwinski M, Tseng TT, Wachi S, Young GB (1999). "Phylogenetic characterization of novel transport protein families revealed by genome analyses." Biochim Biophys Acta 1999;1422(1);1-56. PMID: 10082980

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Zhang07: Zhang N, Chen R, Young N, Wishart D, Winter P, Weiner JH, Li L (2007). "Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS." Proteomics 7(4);484-93. PMID: 17309111

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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