Projection structure of a ClC-type chloride channel at 6.5 Å resolution

Mindell, Joseph A.; Maduke, Merritt; Miller, Christopher; Grigorieff, Nikolaus

doi:10.1038/35051631

Cited by 116 publications

(94 citation statements)

References 16 publications

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“…6 and Table III). This assay, used earlier to study the bacterial chloride channel, EriC [26,27], verified recovery of CFTR channel function with appropriate responses to chloride channel blockers. The more general utility of this assay is also worth comment, since we were able to use it to exclude the possibility of ATP transport via CFTR (Table III).…”

Section: In Vitro Transport By Cftrmentioning

confidence: 65%

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

2004

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The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMPactivated chloride channel. A member of the ATP-binding cassette superfamily of membrane transporters, CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. It is presumed that CFTR couples ATP hydrolysis to channel gating, and as a first step in addressing this issue directly, we have established conditions for purification of biochemical quantities of human CFTR expressed in Sf9 insect cells. Use of an 8-azido-[α 32 P]-ATP binding and vanadate-trapping assay allowed us to devise conditions to preserve CFTR function during purification of a C-terminal His 10 -tagged variant after solubilization with lyso-phosphatidylglycerol (1%) and diheptanolylphosphatidylcholine (0.3%) in the presence of excess phospholipid. Study of purified and reconstituted CFTR showed that it binds nucleotide with an efficiency comparable to that of P-glycoprotein, and that it hydrolyzes ATP at rates sufficient to account for presumed in vivo activity (V Max of 58 ± 5 nmol/min/mg protein, K M (MgATP) of 0.15 mM). In further work, we found that neither nucleotide binding nor ATPase activity were altered by phosphorylation (using Protein Kinase A) or dephosphorylation (with Protein Phosphatase 2B); we also observed inhibition (∼40%) of ATP hydrolysis by reduced glutathione, but not by DTT. To evaluate CFTR function as an anion channel, we introduced an in vitro macroscopic assay based on the equilibrium exchange of proteoliposome-entrapped radioactive tracers. This revealed a CFTRdependent transport of 125 I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [α 32 P]-ATP was observed. We conclude that heterologous expression of CFTR in Sf9 cells can support manufacture and purification of fully functional CFTR. This should aid in further biochemical characterization of this important molecule.The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is the cAMP-activated chloride channel encoded by the gene defective in patients with the disease [1,2]. As a member of the ATP-Binding Cassette (ABC) superfamily of membrane transporters, CFTR shares a conserved architecture consisting of two homologous halves, each containing a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). In CFTR, unlike other ABC transporters, a third domain, termed the regulatory (R) domain, is located between the two half molecules. Current evidence suggests that the TMDs define the CFTR chloride channel, while the NBDs and the R domain mediate channel gating [3][4][5][6][7][8][9][10][11] Although CFTR is glycosylated, there is currently no evidence indicating that the presence of carbohydrate affects CFTR structure or function [12]. Consistent with this presumption, expression of human CFTR in Sf9 insect cells results in appearance of the 140 kD core polypeptide -containing little or no glycosylation -that mediates a newly acquired anion ...

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Section: In Vitro Transport By Cftrmentioning

confidence: 65%

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

2004

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“…More recent work on the cloned CLC channels has fully confirmed this unique architecture: CLC channels are homdimeric proteins with two physically separate (but identical) conduction pathways [Ludewig et al, 1996;Middleton et al, 1996]. Recently a low-resolution projection structure of a bacterial CLC homologue has also confirmed the double-barreled architecture [Mindell et al, 2001]. …”

Section: Clc-1 and The Clc Family Of CL Channelsmentioning

confidence: 79%

“…Probably, a satisfactory understanding of the phenotypic effect of mutations is only possible if an atomicresolution 3D structure is available. The lowresolution structure of the bacterial CLC homologue Eric [Mindell et al, 2001] is not sufficient but it raises the hopes that a high-resolution structure will be available rather soon. Such a structure alone might not be enough to understand immediately the mechanisms of function, but has to be supported by rational mutagenesis.…”

Section: Therapeutic Strategiesmentioning

confidence: 99%

Myotonia caused by mutations in the muscle chloride channel geneCLCN1

2002

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Pure non-syndromic, non-dystrophic myotonia in humans is caused by mutations in the genes coding for the skeletal muscle sodium channel (SCN5A) or the skeletal muscle chloride channel (CLCN1) with similar phenotypes. Chloride-channel myotonia can be dominant (Thomsen-type myotonia) or recessive (Becker-type myotonia). More than 60 myotonia-causing mutations in the CLCN1 gene have been identified, with only a few of them being dominant. A common phenotype of dominant mutations is a dominant negative effect of mutant subunits in mutant-WT heterodimers, causing a large shift of the steady-state open probability voltage-dependence towards more positive, unphysiological voltages. The study of the properties of disease causing mutations has helped in understanding the functional properties of the CLC-1 channel that is part of a nine-member gene family of chloride channels. The large body of knowledge obtained for CLC-1 may also help to better understand the other CLC channels, three of which are also involved in genetic diseases.

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“…In many cases, function and structure are mutually enlightening, but some notable exceptions stand out, of which arguably the most peculiar is CLC-ec1, a bacterial homolog of the widespread CLC family of proteins that move Cl Ϫ ions across membranes in most organisms (5,6). This protein was thought to be a Cl Ϫ channel when initially described (7)(8)(9)(10), but on close electrophysiological scrutiny it turned out to be a Cl Ϫ /H ϩ exchange transporter (11). This surprising result led swiftly to the realization that the entire CLC family is split into channel and transporter subclasses (12)(13)(14)(15); the human genome, for instance, encodes four CLC channels and five CLC transporters.…”

mentioning

confidence: 99%

Ion permeation through a Cl ⁻ -selective channel designed from a CLC Cl ⁻ /H ⁺ exchanger

Jayaram

Accardi

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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148

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The CLC family of Cl ؊ -transporting proteins includes both Cl ؊ channels and Cl ؊ /H ؉ exchange transporters. CLC-ec1, a structurally known bacterial homolog of the transporter subclass, exchanges two Cl ؊ ions per proton with strict, obligatory stoichiometry. Point mutations at two residues, Glu 148 and Tyr 445 , are known to impair H ؉ movement while preserving Cl ؊ transport. In the x-ray crystal structure of CLC-ec1, these residues form putative ''gates'' flanking an ion-binding region. In mutants with both of the gate-forming side chains reduced in size, H ؉ transport is abolished, and unitary Cl ؊ transport rates are greatly increased, well above values expected for transporter mechanisms. Cl ؊ transport rates increase as side-chain volume at these positions is decreased. The crystal structure of a doubly ungated mutant shows a narrow conduit traversing the entire protein transmembrane width. These characteristics suggest that Cl ؊ flux through uncoupled, ungated CLC-ec1 occurs via a channel-like electrodiffusion mechanism rather than an alternating-exposure conformational cycle that has been rendered proton-independent by the gate mutations.

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Projection structure of a ClC-type chloride channel at 6.5 Å resolution

Cited by 116 publications

References 16 publications

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

Myotonia caused by mutations in the muscle chloride channel geneCLCN1

Ion permeation through a Cl ⁻ -selective channel designed from a CLC Cl ⁻ /H ⁺ exchanger

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Projection structure of a ClC-type chloride channel at 6.5 Å resolution

Cited by 116 publications

References 16 publications

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

Myotonia caused by mutations in the muscle chloride channel geneCLCN1

Ion permeation through a Cl − -selective channel designed from a CLC Cl − /H + exchanger

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Ion permeation through a Cl ⁻ -selective channel designed from a CLC Cl ⁻ /H ⁺ exchanger