Structure of a Eukaryotic CLC Transporter Defines an Intermediate State in the Transport Cycle

Feng, Liang; Campbell, Ernest B.; Hsiung, Yichun; MacKinnon, Roderick

doi:10.1126/science.1195230

Cited by 253 publications

(565 citation statements)

References 58 publications

(116 reference statements)

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“…5 A) of the channel membrane domain via conserved histidine and tyrosine residues that are required for channel regulation (14). Conformational changes in the Bateman domain dimer could thus be transduced via the H-I loop to the membrane domain.…”

Section: Discussionmentioning

confidence: 99%

“…Bateman domains in multimeric proteins, including CLCs, dimerize to form a Bateman domain dimer (14)(15)(16)(17)19,20). To determine whether the dimer is required for regulation, we used concatemeric channel constructs generated by fusing two channel monomers C-terminus to N-terminus via a 19-amino-acid linker.…”

Section: Figurementioning

confidence: 99%

“…5 A). The Bateman domains of CLCs dimerize in a unique V-shaped structure (14,15,17). The interface between Bateman domains is formed at a1, b1, and b2 of CBS2 and CBS2 0 , and b3 of CBS1 and CBS1 0 (Fig.…”

Section: Figurementioning

confidence: 99%

“…Each subunit consists of 18 a-helical domains designated A-R (12)(13)(14). Eukaryotic and some prokaryotic CLC proteins also have large cytoplasmic C-termini containing two cystathionine-b-synthase (CBS) domains, termed CBS1 and CBS2, that dimerize to form a Bateman domain (14)(15)(16)(17). The Bateman domains on each CLC subunit interact to form a Bateman domain dimer that contributes to the overall homodimeric channel/transporter architecture (14).…”

Section: Introductionmentioning

confidence: 99%

“…Mutation of a conserved tyrosine residue, Y232, located on a short intracellular loop connecting membrane helices H and I (H-I loop), or a conserved histidine residue, H805, on CBS2 was shown to prevent phosphorylation-induced reductions in channel activity (37,38). The crystal structure of the intact algal CLC, CmCLC, suggests that these two conserved amino acid residues may functionally interact (14). We have proposed that the putative Y232-H805 interaction represents a conserved CLC signal transduction module that couples intracellular and membrane domains (37).…”

Section: Introductionmentioning

confidence: 99%

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Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel

Yamada

Krzeminski

Bozóky

et al. 2016

Biophysical Journal

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Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple a-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-b-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/ Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane a-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structurefunction relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways.

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Section: Discussionmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel

Yamada

Krzeminski

Bozóky

et al. 2016

Biophysical Journal

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Chansporter complexes in cell signaling

Abbott

2017

FEBS Letters

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Ion channels facilitate diffusion of ions across cell membranes for such diverse purposes as neuronal signalling, muscular contraction, and fluid homeostasis. Solute transporters often utilize ionic gradients to move aqueous solutes up their concentration gradient, also fulfilling a wide variety of tasks. Recently, an increasing number of ion channel-transporter (“chansporter”) complexes has been discovered. Chansporter complex formation may overcome what could otherwise be considerable spatial barriers to rapid signal integration and feedback between channels and transporters, the ions and other substrates they transport, and environmental factors to which they must respond. Here, current knowledge in this field is summarized, covering both heterologous expression structure/function findings and potential mechanisms by which chansporter complexes fulfil contrasting roles in cell signalling in vivo.

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An Artificial Single Molecular Channel Showing High Chloride Transport Selectivity and pH‐Responsive Conductance

Huang

Wang

et al. 2023

Angewandte Chemie

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Inspired by the unique structure and function of the natural chloride channel (ClC) selectivity filter, we present herein the design of a ClC‐type single channel molecule. This channel displays high ion transport activity with half‐maximal effective concentration, EC50, of 0.10 μM, or 0.075 mol % (channel molecule to lipid ratio), as determined by fluorescent analysis using lucigenin‐encapsulated vesicles. Planar bilayer lipid membrane conductance measurements indicated an excellent Cl−/K+ selectivity with a permeability ratio P Cl- ${{_{{\rm Cl}{^{- }}}}}$ /P K+ ${{_{{\rm K}{^{+}}}}}$ up to 12.31, which is comparable with the chloride selectivity of natural ClC proteins. Moreover, high anion/anion selectivity (P Cl- ${{_{{\rm Cl}{^{- }}}}}$ /P Br- ${{_{{\rm Br}{^{- }}}}}$ =66.21) and pH‐dependent conductance and ion selectivity of the channel molecule were revealed. The ClC‐like transport behavior is contributed by the cooperation of hydrogen bonding and anion–π interactions in the central macrocyclic skeleton, and by the existence of pH‐responsive terminal phenylalanine residues.

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Structure of a Eukaryotic CLC Transporter Defines an Intermediate State in the Transport Cycle

Cited by 253 publications

References 58 publications

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel

Chansporter complexes in cell signaling

An Artificial Single Molecular Channel Showing High Chloride Transport Selectivity and pH‐Responsive Conductance

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