Crystal structures of a double-barrelled fluoride ion channel

Stockbridge, Randy B.; Kolmakova-Partensky, Ludmila; Shane, Tania; Koide, Akiko; Koide, Shohei; Miller, Christopher; Newstead, Simon

doi:10.1038/nature14981

Cited by 115 publications

(210 citation statements)

References 40 publications

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“…As was proposed on the basis of the initial structure of BEST1, we envisage that the aromatic side chains of phenylalanines F80 and F84 within the neck interact with anions flowing through the pore via encircling rings of anion-quadrupole interactions (16). In accord with this hypothesis, anion-quadrupole interactions have been observed in a bacterial fluoride channel and mutation of the interacting phenylalanine residues to isoleucine also reduced ion flux through that channel (32). Anion-quadrupole interactions mediated by the phenylalanines may reduce the energy barriers for Cl − and other anions to pass through the neck relative to other hydrophobic amino acids, but these interactions must not be the determining factor for charge selectivity in eukaryotic bestrophin channels, because their removal has almost no effect on anion-vs.-cation selectivity.…”

Section: +mentioning

confidence: 55%

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

Vaisey

Miller

Long

2016

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

135

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Cytoplasmic calcium (Ca 2+ ) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca 2+ -independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca 2+ activation and ion selectivity. A "Ca 2+ clasp" within the channel's intracellular region acts as a sensor of cytoplasmic Ca 2+ . Alanine substitutions within a hydrophobic "neck" of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca 2+. We conclude that the primary function of the neck is as a "gate" that controls chloride permeation in a Ca 2+ -dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel's ion selectivity. We find that mutation of a cytosolic "aperture" of the pore does not perturb the Ca 2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single "selectivity filter," in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.calcium-activated chloride channel | CaCC | ion selectivity | channel gating | ion channel biophysics E ukaryotic bestrophin proteins constitute a family of Ca 2+

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Section: +mentioning

confidence: 55%

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

Vaisey

Miller

Long

2016

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

135

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“…These residues are near the sodium binding site and some of them coordinate a putative sodium ion using four backbone carbonyl groups [14]. The general sequence is ooF II ooF II , where o is either S or T, and F II is F in Pore II but is not a conserved residue in Pore I (see above).…”

Section: Resultsmentioning

confidence: 99%

Fluoride export (FEX) proteins from fungi, plants and animals are 'single barreled' channels containing one functional and one vestigial ion pore

et al. 2017

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The fluoride export protein (FEX) in yeast and other fungi provides tolerance to fluoride (F-), an environmentally ubiquitous anion. FEX efficiently eliminates intracellular fluoride that otherwise would accumulate at toxic concentrations. The FEX homolog in bacteria, Fluc, is a ‘double-barreled’ channel formed by dimerization of two identical or similar subunits. FEX in yeast and other eukaryotes is a monomer resulting from covalent fusion of the two subunits. As a result, both potential fluoride pores are created from different parts of the same protein. Here we identify FEX proteins from two multicellular eukaryotes, a plant Arabidopsis thaliana and an animal Amphimedon queenslandica, by demonstrating significant fluoride tolerance when these proteins are heterologously expressed in the yeast Saccharomyces cerevisiae. Residues important for eukaryotic FEX function were determined by phylogenetic sequence alignment and functional analysis using a yeast growth assay. Key residues of the fluoride channel are conserved in only one of the two potential fluoride-transporting pores. FEX activity is abolished upon mutation of residues in this conserved pore, suggesting that only one of the pores is functional. The same topology is conserved for the newly identified FEX proteins from plant and animal. These data suggest that FEX family of fluoride channels in eukaryotes are ‘single-barreled’ transporters containing one functional pore and a second non-functional vestigial remnant of a homologous gene fusion event.

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“…DARPins (32)(33)(34) and monobodies (35,36) have been used to solve x-ray structures of such challenging targets as membrane transporters. Nanobodies and SPARs share many of the same advantages, such as small size, single domain composition, and the ease of producing recombinant proteins.…”

Section: Nanobodies Versus Specific Binding Proteins Designed On Non-mentioning

confidence: 99%

Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

Dmitriev

Lutsenko

Muyldermans

2016

Journal of Biological Chemistry

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Nanobodies are the recombinant antigen-recognizing domains of the minimalistic heavy chain-only antibodies produced by camels and llamas. Nanobodies can be easily generated, effectively optimized, and variously derivatized with standard molecular biology protocols. These properties have triggered the recent explosion in the nanobody use in basic and clinical research. This review focuses on the emerging use of nanobodies for understanding and monitoring protein dynamics on the scales ranging from isolated protein domains to live cells, from nanoseconds to hours. The small size and high solubility make nanobodies uniquely suited for studying protein dynamics by NMR. The ability to produce conformation-sensitive nanobodies in cells enables studies that link structural dynamics of a target protein to its cellular behavior. The link between in vitro and in-cell dynamics, afforded by nanobodies, brings the analysis of such important events as receptor signaling, membrane protein trafficking, and protein interactions to the next level of resolution.

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Crystal structures of a double-barrelled fluoride ion channel

Cited by 115 publications

References 40 publications

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

Fluoride export (FEX) proteins from fungi, plants and animals are 'single barreled' channels containing one functional and one vestigial ion pore

Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

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