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Cited by 70ATP Synthase and ATPases ResearchIon Transport and Channel RegulationIon channel regulation and function

The DOI matches its Crossref record; 102 of the 102 references it cites were found in a registry or an index; 1 of the 169 sources its claims cite were checked for support.

Membrane Exporters of Fluoride Ion

Open access
McIlwain, Benjamin C., Ruprecht, Michal T., Stockbridge, Randy B.·2021·Annual Review of Biochemistry
DOI 10.1146/annurev-biochem-071520-112507PMC PMC8217344
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Abstract

Microorganisms contend with numerous and unusual chemical threats and have evolved a catalog of resistance mechanisms in response. One particularly ancient, pernicious threat is posed by fluoride ion (F − ), a common xenobiotic in natural environments that causes broad-spectrum harm to metabolic pathways. This review focuses on advances in the last ten years toward understanding the microbial response to cytoplasmic accumulation of F − , with a special emphasis on the structure and mechanisms of the proteins that microbes use to export fluoride: the CLC F family of F − /H + antiporters and the Fluc/FEX family of F − channels.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

This review summarizes how microorganisms export fluoride ion via two unrelated membrane protein families: CLCF F−/H+ antiporters and Fluc/FEX F− channels. It covers environmental fluoride exposure, weak-acid accumulation and toxicity, and the structures and proposed mechanisms of both exporter families.

Key findings

  • CLCFs are secondary active F−/H+ antiporters that export one F− in exchange for one H+, with unitary transport rates of ~400–1000 ions per second and high F− selectivity over Cl−; Cl− permeation is not observed unless a high membrane potential is applied.
  • Fluc/FEX proteins are electrodiffusive, constitutively open F− channels with conductance corresponding to ~10^5–6 ions/sec and F−/Cl− selectivity of at least 10,000-fold.
  • Fluoride toxicity arises from weak acid accumulation: HF (pKa = 3.4) crosses membranes, and at external pH 5.5 with cytoplasmic pH 7.5, intracellular fluoride accumulates 100-fold, reaching millimolar concentrations; F− inhibits metalloenzymes with inhibitory constants in the hundreds of micromolar range.
  • CLCF structures show central (Fcen) and external (Fext) anion binding sites, with Fcen coordinated by Y396 and M79 and Fext by T320 and backbone amides G116–G119; the gating glutamate E118 drives a transport cycle. Fluc structures propose F0, F1, and F2 sites, a polar track, a phenylalanine box (F82/F85), and a conserved glutamate E88, but the basis of Fluc selectivity remains unresolved.

Methods

  • Narrative review of advances over the last ten years in understanding microbial fluoride export, with no new primary experimental data reported.
  • Synthesizes environmental, biochemical, structural, and functional literature on fluoride toxicity, fluoride riboswitches, CLCF antiporters, and Fluc/FEX channels, including X-ray crystallography, electrophysiology, mutagenesis, and transport assays from cited studies.
  • Uses figures and an equation to illustrate weak acid accumulation and proposed transport mechanisms.

Limitations

  • The paper does not state explicit limitations. As a review, it relies on published studies and does not present new primary data.
  • The text makes evident that the features responsible for the Fluc channels’ striking 10,000-fold F−/Cl− selectivity have not yet been identified; of dozens of mutations, none allowed detectable throughput of any other anion.
  • Some Fluc structural and functional data are cited as manuscript in preparation, including the F0 site identification and MTSES inhibition experiments.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1146/annurev-biochem-071520-112507

Cites

102 of 102 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

1 of 169 cited sources checked · 1 supported · 0 flagged

  • Interpretationcitation

    Expression of F− export proteins in the plasma membrane undermines weak acid accumulation of fluoride.

    “By providing a route for accumulating F− to exit the cell, expression of these F− export proteins in the plasma membrane simply undermines weak acid accumulation of fluoride (1, 14, 25, 26).”
    Quote in the paperSubstantial claim1 of 4 sources checked
    • cites [1] Stockbridge RB, Lim HH, Otten R, Williams C, Shane T, 2012. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci U S A 109: 15289– 94 22949689 PMC3458365 source
    • cites [14] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. The Journal of General Physiology 144: 257– 61 25156118 PMC4144673 source
    • cites [25] Baker JL, Sudarsan N, Weinberg Z, Roth A, Stockbridge RB, Breaker RR. 2012. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335: 233– 5 22194412 PMC4140402 source
    • cites [26] Li S, Smith KD, Davis JH, Gordon PB, Breaker RR, Strobel SA. 2013. Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins. Proceedings of the National Academy of Sciences 110: 19018– 23 PMC3839697 24173035 source
  • Premisecitation

    For the Flucs, the positive-outside electrical potential thermodynamically favors anion expulsion.

    “For the Flucs, the positive-outside electrical potential thermodynamically favors anion expulsion (2).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Stockbridge RB, Robertson JL, Kolmakova-Partensky L, Miller C. 2013. A family of fluoride-specific ion channels with dual-topology architecture. Elife 2: e01084 23991286 PMC3755343 source
    • Rests on an assumption: The proton motive force is maintained by metabolizing cells.
  • Premisereasoning

    The CLCF and Fluc/FEX proteins bind F− without the aid of any metal ion.

    “Moreover, they draw a stark contrast with most cases of fluoride complexation by biological macromolecules, in that they bind F− without the aid of any metal ion.”
    • Also stated in: § CHALLENGES IN FLUORIDE RECOGNITION · plain
      “And in contrast to the majority of F− binding macromolecules, including those shown in Figure 2a–d, neither membrane exporter employs metal ions to bind or transport F−, providing rare examples of biological F− coordination without intervening metals.”
    Quote in the paperSubstantial claim
  • Interpretationreasoning

    The common challenge shared by the CLCFs and the Flucs is to rapidly export F− with high specificity.

    “The common challenge shared – and met – by the CLCFs and the Flucs is to rapidly export F−, but to do so with high specificity.”
    Quote in the paperSubstantial claim
  • Premisecitation

    In pyrophosphatase, F− displaces the hydrolytic water in the coordination sphere of the enzyme-bound Mg2+, acting as a transition state analogue of the activated water molecule.

    “For example, in pyrophosphatase, F− displaces the hydrolytic water in the coordination sphere of the enzyme-bound Mg2+, acting as a transition state analogue of the activated water molecule(17, 18) (Figure 2a).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [17] Samygina VR, Moiseev VM, Rodina EV, Vorobyeva NN, Popov AN, 2007. Reversible inhibition of Escherichia coli inorganic pyrophosphatase by fluoride: trapped catalytic intermediates in cryo-crystallographic studies. J Mol Biol 366: 1305– 17 17196979 source
    • cites [18] Heikinheimo P, Tuominen V, Ahonen AK, Teplyakov A, Cooperman BS, 2001. Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase. Proceedings of the National Academy of Sciences of the United States of America 98: 3121– 26 11248042 PMC30617 source
  • It may state two separate claims as one. 55
  • Probably not a substantial claim. 15

  • It may state two separate claims as one.

    Premisecitation

    The energetic cost to dehydrate fluoride is 111 kcal/mol, approximately 30 kcal/mol more costly than dehydration of Cl−.

    “This is reflected in the high energetic cost to dehydrate fluoride, 111 kcal/mol, approximately 30 kcal/mol more costly than dehydration of Cl− (29).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] Marcus Y 1994. A Simple Empirical-Model Describing the Thermodynamics of Hydration of Ions of Widely Varying Charges, Sizes, and Shapes. Biophysical Chemistry 51: 111– 27 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Human activity further increases ambient F− concentrations over environmental levels.

    “In some areas, human activity further increases ambient F− concentrations over environmental levels.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [7] Division of Oral Health: My Water’s Fluoride web application. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention (CDC), National Center for Chronic Disease Prevention and Health Promotion, Atlanta, GA, 2016. Available at http://www.cdc.gov/oralhealth/.
    • cites [4] Weinstein LH, Davison A. 2004. Fluorides in the environment: effects on plants and animals. Cambridge, MA: CABI Publishing
    • cites [8] Johnston NR, Strobel SA. 2020. Principles of fluoride toxicity and the cellular response: a review. Archives of Toxicology 94: 1051– 69 32152649 PMC7230026 source

  • It may state two separate claims as one.

    Premisecitation

    Fluoride ion (F−) is a common xenobiotic in natural environments that causes broad-spectrum harm to metabolic pathways.

    “One particularly ancient, pernicious threat is posed by fluoride ion (F−), a common xenobiotic in natural environments that causes broad-spectrum harm to metabolic pathways.”
    • Also stated in: § F− inhibition of biomolecules · plain
      “Once it has breached the membrane and accumulated within the cell, fluoride is detrimental to biological systems because of its ability to act as substrate or transition state analogues for a host of metalloenzymes involved in the most fundamental biological processes, ranging from glycolysis to nitrogen fixation to macromolecule synthesis(13, 15, 16).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [13] Marquis RE, Clock SA, Mota-Meira M. 2003. Fluoride and organic weak acids as modulators of microbial physiology. FEMS Microbiol Rev 26: 493– 510 12586392 source
    • cites [15] Adamek E, Pawlowska-Goral K, Bober K. 2005. In vitro and in vivo effects of fluoride ions on enzyme activity. Ann Acad Med Stetin 51: 69– 85 16519100
    • cites [16] Johnston NR, Strobel SA. 2019. Nitrate and Phosphate Transporters Rescue Fluoride Toxicity in Yeast. Chemical Research in Toxicology 32: 2305– 19 31576749 PMC7316403 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Fluoridation of municipal water supplies in the United States sets the F− concentration of drinking water to ~40 μM in many locations.

    “Fluoridation of municipal water supplies in the United States sets the F− concentration of drinking water to ~40 μM in many locations (7)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Division of Oral Health: My Water’s Fluoride web application. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention (CDC), National Center for Chronic Disease Prevention and Health Promotion, Atlanta, GA, 2016. Available at http://www.cdc.gov/oralhealth/.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    F− concentrations are higher in areas affected by industrial pollution or by use of fluoride-contaminated fertilizers, and in the oral microbiome, toothpaste has ~70 mM fluoride.

    “and F− concentrations are higher still in areas affected by industrial pollution or by use of fluoride-contaminated fertilizers; and in niches such as the oral microbiome, where exposure to fluoride for dental hygiene – the concentration of fluoride ion is ~70 mM in toothpaste – is routine(4, 8).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Weinstein LH, Davison A. 2004. Fluorides in the environment: effects on plants and animals. Cambridge, MA: CABI Publishing
    • cites [8] Johnston NR, Strobel SA. 2020. Principles of fluoride toxicity and the cellular response: a review. Archives of Toxicology 94: 1051– 69 32152649 PMC7230026 source

  • It may state two separate claims as one.

    Interpretationcitation

    In microbes that lack any F− export pathway, intracellular F− accumulates according to Eq. 1; for a microbe in a modestly acidic niche of pH 5.5 that maintains a cytoplasmic pH of 7.5, the intracellular fluoride will accumulate 100-fold in the cytoplasm compared to environmental levels, reaching millimolar concentrations.

    “In microbes that lack any F− export pathway, intracellular F− accumulates according to this simple scheme: (Eq. 1)F−in F−out =H+out H+in so that for a microbe in a modestly acidic niche of pH 5.5 that maintains a cytoplasmic pH of 7.5, the intracellular fluoride will accumulate 100-fold in the cytoplasm compared to environmental levels, reaching millimolar concentrations (14).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [14] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. The Journal of General Physiology 144: 257– 61 25156118 PMC4144673 source

  • It may state two separate claims as one.

    Premisecitation

    Other anions like chloride (Cl−) are more abundant in the cytoplasm (10-100 mM in E. coli), and even low levels of uncontrolled leak would disrupt the membrane potential with catastrophic consequences for processes that rely on the electrical potential.

    “Other anions like chloride (Cl−) are more abundant in the cytoplasm (10–100 mM in E. coli(27)), and even low levels of uncontrolled leak would disrupt the membrane potential with catastrophic consequences for processes that rely on the electrical potential, including ATP synthesis, secondary active transport, and motility.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27] Schultz SG, Wilson NL, Epstein W. 1962. Cation transport in Escherichia coli. II. Intracellular chloride concentration. J Gen Physiol 46: 159– 66 13909522 PMC2195246 source

  • It may state two separate claims as one.

    Premisenone given

    CLCF and Fluc are unrelated by sequence, structure, and mechanism, but their physiological roles overlap; most bacteria have one or the other, but not both.

    “Although CLCF and Fluc are unrelated by sequence, structure, and mechanism, their physiological roles overlap, and most bacteria have one or the other, but not both.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    HF is a weak acid (pKa = 3.4) and readily crosses the plasma membrane.

    “The conjugate acid HF is a weak acid (pKa = 3.4), and because HF is small and uncharged, it readily crosses the plasma membrane (9, 10).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [9] Barbier O, Arreola-Mendoza L, Del Razo LM. 2010. Molecular mechanisms of fluoride toxicity. Chem Biol Interact 188: 319– 33 20650267 source
    • cites [10] Orabi EA, Faraldo-Gomez JD. 2020. A New Molecular-Mechanics Model for Simulations of Hydrogen Fluoride in Chemistry and Biology. J Chem Theory Comput PMC8431972 32615034 source

  • It may state two separate claims as one.

    Interpretationcitation

    Aluminium fluoride inhibition of phosphoryl group transfer enzymes is a germane biological phenomenon because aluminium is found at physiologically relevant concentrations in soils.

    “Since aluminium is also found at physiologically relevant concentrations in soils, aluminium fluoride inhibition of phosphoryl group transfer enzymes is a germane biological phenomenon (4).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Weinstein LH, Davison A. 2004. Fluorides in the environment: effects on plants and animals. Cambridge, MA: CABI Publishing

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisenone given

    Na+/H+ antiporter genes frequently co-occur with fluoride riboswitches.

    “Na+/H+ antiporter genes also frequently co-occur with fluoride riboswitches.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    Inner sphere fluoride-water interactions are dominated by classical hydrogen bonds, which are more tightly structured and closely held.

    “Theoretical treatments of halides show that inner sphere fluoride-water interactions are dominated by classical hydrogen bonds(37, 38), which are more tightly structured and closely held(33).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [37] Muralidharan A, Pratt LR, Chaudhari MI, Rempe SB. 2018. Quasi-Chemical Theory with Cluster Sampling from Ab Initio Molecular Dynamics: Fluoride (F(−)) Anion Hydration. J Phys Chem A 122: 9806– 12 30475612 source
    • cites [38] Muralidharan A, Pratt LR, Chaudhari MI, Rempe SB. 2019. Quasi-chemical theory for anion hydration and specific ion effects: Cl-(aq) vs. F-(aq). Chemical Physics Letters: X 4: 100037 source
    • cites [33] Bostick DL, Brooks CL. 2009. Statistical Determinants of Selective Ionic Complexation: Ions in Solvent, Transport Proteins, and Other “Hosts”. Biophysical Journal 96: 4470– 92 19486671 PMC2894556 source

  • It may state two separate claims as one.

    Premisereasoning

    Neither membrane exporter employs metal ions to bind or transport F−, providing rare examples of biological F− coordination without intervening metals.

    “And in contrast to the majority of F− binding macromolecules, including those shown in Figure 2a–d, neither membrane exporter employs metal ions to bind or transport F−, providing rare examples of biological F− coordination without intervening metals.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisecitation

    The charged species (H+ and F−) cannot diffuse back across the membrane out of the cell, and thus accumulate, a process known as weak acid accumulation.

    “These charged species cannot diffuse back across the membrane out of the cell, and thus accumulate, a process known as weak acid accumulation, or ion-trapping(13).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Marquis RE, Clock SA, Mota-Meira M. 2003. Fluoride and organic weak acids as modulators of microbial physiology. FEMS Microbiol Rev 26: 493– 510 12586392 source

  • It may state two separate claims as one.

    Interpretationreasoning

    F− has a higher charge density than Cl− and is an especially strong hydrogen bond acceptor.

    “As such, F− also has a higher charge density than Cl−, and is therefore an especially strong hydrogen bond acceptor.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisecitation

    Fluoride exporters from the CLCF and Fluc families are not necessarily associated with a riboswitch; some, including the E. coli Fluc channel, are instead constitutively expressed.

    “Fluoride exporters from the CLCF and Fluc families are not necessarily associated with a riboswitch(1, 25); some, including the E. coli Fluc channel, are instead constitutively expressed (14).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [1] Stockbridge RB, Lim HH, Otten R, Williams C, Shane T, 2012. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci U S A 109: 15289– 94 22949689 PMC3458365 source
    • cites [25] Baker JL, Sudarsan N, Weinberg Z, Roth A, Stockbridge RB, Breaker RR. 2012. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335: 233– 5 22194412 PMC4140402 source
    • cites [14] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. The Journal of General Physiology 144: 257– 61 25156118 PMC4144673 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Natural environmental fluoride concentrations range from 30-80 μM in oceans, and from 10 μM - 400 μM in ground and surface waters.

    “Natural environmental fluoride concentrations vary widely depending on the local geology, and range from 30–80 μM in oceans, and from 10 μM - 400 μM in ground and surface waters(4, 6).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Weinstein LH, Davison A. 2004. Fluorides in the environment: effects on plants and animals. Cambridge, MA: CABI Publishing
    • cites [6] Windom HL. 1971. Fluoride Concentration in Coastal and Estuarine Waters of Georgia. Limnology and Oceanography 16: 806-& source

  • It may state two separate claims as one.

    Premisecitation

    F− is the smallest of the halides, with a radius about half an Angstrom smaller than Cl−.

    “F− is the smallest of the halides, with a radius about half an Angstrom smaller than Cl− (28).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Shannon RD. 1976. Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides. Acta Crystallogr A 32: 751– 67 source

  • It may state two separate claims as one.

    Interpretationreasoning

    Fluoride recognition by proteins is challenging due to the close size of prominent biological competitors and the especially high free energy of dehydration.

    “Thus, fluoride recognition by proteins is challenging due to the close size of prominent biological competitors and the especially high free energy of dehydration.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    Although CLCFs share many mechanistic features with the large CLC family of Cl− transporters, both the anion binding sites and the interactions between fluoride and the gating glutamate have been optimized for F− transduction.

    “Thus, although the proteins CLCFs share many mechanistic features with the large CLC family of Cl− transporters, both the anion binding sites and the interactions between the fluoride and the gating glutamate have been optimized for F− transduction.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisecitation

    Breaks in these helices position the positive dipoles of the N-terminal ends near the central dehydrated anion-binding region and provide backbone amides to coordinate transient anions.

    “Breaks in these helices position the positive dipoles of the N-terminal ends of the helices near the central dehydrated anion-binding region of the protein, and also provide backbone amides to coordinate the transient anions (Figure 3d).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Last NB, Stockbridge RB, Wilson AE, Shane T, Kolmakova-Partensky L, 2018. A CLC-type F(−)/H(+) antiporter in ion-swapped conformations. Nat Struct Mol Biol PMC6044475 29941917 source

  • It may state two separate claims as one.

    Premisecitation

    The Fcen site sits at the apex of the cytoplasmic vestibule, exposed to water and coordinated by protein sidechains in a manner reminiscent of other CLCs.

    “The first of the fluoride binding sites, Fcen, sits at the apex of the cytoplasmic vestibule, where it is exposed to water and coordinated by protein sidechains in a manner reminiscent of other CLCs.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Last NB, Stockbridge RB, Wilson AE, Shane T, Kolmakova-Partensky L, 2018. A CLC-type F(−)/H(+) antiporter in ion-swapped conformations. Nat Struct Mol Biol PMC6044475 29941917 source

  • It may state two separate claims as one.

    Premisecitation

    Knockouts of genomic Fluc/FEX proteins increase fluoride sensitivity of bacteria including E. coli, B. subtilis, and S. sanguinis, and fungi including S. cerevisiae, C. albicans, N. crassa, and A. fumigatus.

    “Knockouts of genomic Fluc/FEX proteins increase fluoride sensitivity of bacteria including Escherichia coli(14, 25), Bacillus subtilis(25, 64), and oral bacterium Streptococcus sanguinis(49), and fungi including Saccharomyces cerevisiae(3, 26), Candida albicans(26), Neurospora crassa(26), and Aspergillus fumigatus(65).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [14] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. The Journal of General Physiology 144: 257– 61 25156118 PMC4144673 source
    • cites [25] Baker JL, Sudarsan N, Weinberg Z, Roth A, Stockbridge RB, Breaker RR. 2012. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335: 233– 5 22194412 PMC4140402 source
    • cites [64] Dong W, Setlow P. 2019. Fluoride movement into and out of Bacillus spores and growing cells and effects of fluoride accumulation on spore properties. J Appl Microbiol 126: 503– 15 30430725 source

  • It may state two separate claims as one.

    Premisecitation

    A sidechain hydroxyl from Y396 coordinates the fluoride, an interaction with precedent in Cl− transporters.

    “In an interaction that has precedent in the Cl− transporters (42), a sidechain hydroxyl from Y396 coordinates the fluoride.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] 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: 287– 94 11796999 source

  • It may state two separate claims as one.

    Premisecitation

    A metabolizing cell maintains a negative-inside membrane potential, so the electrical potential favors anion expulsion.

    “Second, a metabolizing cell maintains a negative-inside membrane potential, so that the electrical potential favors anion expulsion(14).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [14] Ji C, Stockbridge RB, Miller C. 2014. Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. The Journal of General Physiology 144: 257– 61 25156118 PMC4144673 source

  • It may state two separate claims as one.

    Premisecitation

    Each monomer contains the necessary machinery for ion permeation and proton-coupling, and if mutations force the protein into a monomeric state, the lone subunits are capable of functioning independently.

    “Each monomer contains the necessary machinery for ion permeation and proton-coupling, and if mutations are introduced that force the protein into a monomeric state, the lone subunits are capable of functioning independently(51).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] Last NB, Miller C. 2015. Functional Monomerization of a ClC-Type Fluoride Transporter. J Mol Biol 427: 3607– 12 26449639 PMC4847941 source

  • It may state two separate claims as one.

    Interpretationreasoning

    The high F− selectivity of CLCFs is unique among CLCs and makes physiological sense because fluoride export is the only described context in which a CLC must discriminate against monovalent anions more prevalent than the substrate.

    “Such selectivity is unique among CLCs, but makes good physiological sense: fluoride export is the only yet-described context in which a CLC must discriminate against molovalent anions that are more prevalent than the substrate in the biological milieu.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Small molecule hosts designed for fluoride recognition commonly utilize between four and six hydrogen bond donors.

    “small molecule hosts designed for fluoride recognition commonly utilize between four and six hydrogen bond donors (32).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Cametti M, Rissanen K. 2013. Highlights on contemporary recognition and sensing of fluoride anion in solution and in the solid state. Chem Soc Rev 42: 2016– 38 23188119 source

  • It may state two separate claims as one.

    Interpretationreasoning

    There are a number of physicochemical properties that membrane transport proteins could exploit to differentiate F− from Cl−, including the preference of fluoride for fewer ligands and a tighter coordination shell, its greater strength as a hydrogen bond acceptor, and its unique ability to share protons with pKa-matched protonatable sidechains.

    “But there are also a number of physicochemical properties that membrane transport proteins could exploit to differentiate F− from Cl−, including the preference of fluoride for fewer ligands and a tighter coordination shell, its greater strength as a hydrogen bond acceptor, and its unique ability to share protons with pKa-matched protonatable sidechains.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    The transporters harness the transmembrane proton gradient to export one F− in exchange for one H+, and the unitary transport rates of different characterized homologues range from ~400-1000 ions per second.

    “The transporters harness the transmembrane proton gradient to export one F− in exchange for one H+, and the unitary transport rates of different characterized homologues ranges from ~400–1000 ions per second (1, 50).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [1] Stockbridge RB, Lim HH, Otten R, Williams C, Shane T, 2012. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci U S A 109: 15289– 94 22949689 PMC3458365 source
    • cites [50] Brammer AE, Stockbridge RB, Miller C. 2014. F−/Cl− selectivity in CLCF-type F−/H+ antiporters. J Gen Physiol 144: 129– 36 25070431 PMC4113899 source

  • It may state two separate claims as one.

    Premisecitation

    Representatives with and without riboswitches share basic transport properties, and the proteins in this clade were renamed CLCFs.

    “Not all proteins in this clade are associated with a riboswitch, but representatives with and without riboswitches share basic transport properties, and the proteins in this clade were thus renamed CLCFs(1).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Stockbridge RB, Lim HH, Otten R, Williams C, Shane T, 2012. Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci U S A 109: 15289– 94 22949689 PMC3458365 source

  • It may state two separate claims as one.

    Premisecitation

    Mutagenesis experiments showed that M79 contributes to F−/Cl− selectivity in CLCF proteins, and conversion to asparagine reduces selectivity in a close homologue of the E. casseliflavus protein.

    “Mutagenesis experiments showed that this methionine contributes to F−/Cl− selectivity in the CLCF proteins, and conversion to the straightforwardly polar asparagine reduces selectivity in a close homologue of the E. casseliflavus protein(50).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [50] Brammer AE, Stockbridge RB, Miller C. 2014. F−/Cl− selectivity in CLCF-type F−/H+ antiporters. J Gen Physiol 144: 129– 36 25070431 PMC4113899 source

  • It may state two separate claims as one.

    Premisecitation

    The α-helices are tilted with respect to the membrane, defining electropositive internal and external aqueous vestibules that are 6-8 Å deep.

    “In accordance with their role in anion transport, the a-helices are tilted with respect to the membrane, defining electropositive internal and external aqueous vestibules that are 6–8 Å deep (Figure 3c).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Last NB, Stockbridge RB, Wilson AE, Shane T, Kolmakova-Partensky L, 2018. A CLC-type F(−)/H(+) antiporter in ion-swapped conformations. Nat Struct Mol Biol PMC6044475 29941917 source

  • It may state two separate claims as one.

    Premisecitation

    E118 is a critical proton carrier that faces the external solution in the wildtype E. casseliflavus CLCF structure.

    “In addition to the fluoride-coordinating residues, a final critical component of the transport machinery is the proton carrier, E118, which faces the external solution in the structure of the wildtype E. casseliflavus CLCF.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [52] Last NB, Stockbridge RB, Wilson AE, Shane T, Kolmakova-Partensky L, 2018. A CLC-type F(−)/H(+) antiporter in ion-swapped conformations. Nat Struct Mol Biol PMC6044475 29941917 source

  • It may state two separate claims as one.

    Premisecitation

    The coordination sphere of larger halides is more flexible, with both hydrogen bond and dipole-dominated configurations occurring.

    “Their coordination sphere is more flexible, and both hydrogen bond and dipole-dominated configurations occur(38).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Muralidharan A, Pratt LR, Chaudhari MI, Rempe SB. 2019. Quasi-chemical theory for anion hydration and specific ion effects: Cl-(aq) vs. F-(aq). Chemical Physics Letters: X 4: 100037 source

  • It may state two separate claims as one.

    Premisecitation

    The fluoride channel family encompasses a topologically diverse set of proteins that provide an unusual glimpse into evolutionary processes in membrane transport proteins.

    “The fluoride channel family encompasses a topologically diverse set of proteins that provide an unusual glimpse into evolutionary processes in membrane transport proteins(68) (Figure 5a).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [68] Macdonald CB, Stockbridge RB. 2017. A topologically diverse family of fluoride channels. Curr Opin Struct Biol 45: 142– 49 28514705 PMC5612848 source

  • It may state two separate claims as one.

    Interpretationreasoning

    Deprotonated E118 follows the Fcen ➔ Fext ➔ external vestibule anion permeation route (stage 4), pushing a single F− ahead of it, and transits back to the 'up' position in the extracellular vestibule.

    “Deprotonated E118 is proposed to follow the Fcen ➔ Fext ➔ external vestibule anion permeation route (stage 4), pushing a single F− ahead of it, such that E118 transits back to the ‘up’ position in the extracellular vestibule.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisecitation

    Flucs are perhaps the most selective ion channels known; even low levels of Cl− transport are not observed, placing the F−/Cl− selectivity at a minimum of 10,000-fold.

    “Accordingly, the Flucs are perhaps the most selective ion channels known; even low levels of Cl− transport are not observed, placing the F−/Cl− selectivity at a minimum of 10,000-fold(2).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Stockbridge RB, Robertson JL, Kolmakova-Partensky L, Miller C. 2013. A family of fluoride-specific ion channels with dual-topology architecture. Elife 2: e01084 23991286 PMC3755343 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    A number of Cl− transporting CLCs have been structurally characterized, providing a deep mechanistic basis for understanding the fluoride riboswitch-associated CLCs.

    “A number of Cl− transporting CLCs have been structurally characterized, providing a deep mechanistic basis for understanding the fluoride riboswitch-associated CLCs (42–46).”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [42] 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: 287– 94 11796999 source
    • cites [43] 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: 11194– 9 18678918 PMC2516207 source
    • cites [44] Feng L, Campbell EB, Hsiung Y, MacKinnon R. 2010. Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle. Science 330: 635– 41 20929736 PMC3079386 source
    • cites [45] Park E, Campbell EB, MacKinnon R. 2017. Structure of a CLC chloride ion channel by cryo-electron microscopy. Nature 541: 500– 05 28002411 PMC5576512 source
    • cites [46] Park E, MacKinnon R. 2018. Structure of the CLC-1 chloride channel from Homo sapiens. Elife 7 PMC6019066 29809153 source

  • It may state two separate claims as one.

    Interpretationreasoning

    The excursions of this single sidechain carry a H+ to the intracellular side and then electrostatically push a F− to the extracellular side, providing a structural explanation for the 1-to-1 F−/H+ exchange stoichiometry.

    “Thus, the excursions of this single sidechain carries a H+ to the intracellular side, then electrostatically pushes a F− to the extracellular side, providing a structural explanation for the 1-to-1 F−/H+ exchange stoichiometry.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationcitation

    The participation of methionine in fluoride coordination is surprising because hard bases like F− generally prefer coordination by hard acids like asparagine or serine.

    “The methionine is a somewhat surprising participant in fluoride coordination, since, in general, hard bases (like F−) prefer coordination by hard acids, like conventional H-bond donors asparagine or serine(53).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] Pearson RG. 1966. Acids and bases. Science 151: 172– 7 17746330 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Canonical CLC proteins are not very selective among anions and also permeate other halides and pseudohalides like iodide, bromide, or nitrate.

    “Although Cl− is the namesake of the family, and in most cases the physiological ion, the canonical CLC proteins are not very selective among anions and also permeate other halides and pseudohalides like iodide, bromide, or nitrate(41).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] 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: 691– 9 16949616 source

  • It may state two separate claims as one.

    Premisecitation

    In CLC channels, the equivalent glutamate gates the conduction pathway depending on its protonation state, and in CLC transporters, the glutamate participates in transport of the permeant H+.

    “In the CLC channels, the equivalent glutamate gates the conduction pathway depending on its protonation state(54), and in the CLC transporters, the glutamate participates in transport of the permeant H+(55–57).”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [54] Lisal J, Maduke M. 2008. The ClC-0 chloride channel is a ‘broken’ Cl−/H+ antiporter. Nat Struct Mol Biol 15: 805– 10 18641661 PMC2559860 source
    • cites [55] Feng L, Campbell EB, MacKinnon R. 2012. Molecular mechanism of proton transport in CLC Cl−/H+ exchange transporters. Proc Natl Acad Sci U S A 109: 11699– 704 22753511 PMC3406864 source
    • cites [56] Dutzler R, Campbell EB, MacKinnon R. 2003. Gating the selectivity filter in ClC chloride channels. Science 300: 108– 12 12649487 source
    • cites [57] Chavan TS, Cheng RC, Jiang T, Mathews II, Stein RA, 2020. A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl(−)/H(+) transport cycle. Elife 9 PMC7253180 32310757 source

  • It may state two separate claims as one.

    Premisecitation

    S83C and S83T are incompetent for F− transport.

    “These positions are sensitive to mutagenesis: S83C and S83T are incompetent for F− transport, and the double S83A/T84A mutant exhibits currents just 1% of wildtype (B.C. McIlwain, R. Gundepudi, B.B. Koff, and R.B. Stockbridge, manuscript in preparation).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites B.C. McIlwain, R. Gundepudi, B.B. Koff, and R.B. Stockbridge, manuscript in preparation

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    The proposed fluoride binding sites in Fluc may be under-coordinated, with few protein ligands (three or four, assuming limited sidechain rearrangement) in order to exclude Cl−.

    “The proposed fluoride binding sites in Fluc may be “under-coordinated,” with few protein ligands (three or four, assuming limited sidechain rearrangement) in order to exclude Cl−.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Fluoride requires the smallest number of coordinating ligands of any anion.
    • The authors state this as an assumption: Sidechain rearrangement is limited.

  • It may state two separate claims as one.

    Premiseown data

    The channel adopts a symmetrical hourglass shape with wide aqueous vestibules on both sides of the membrane.

    “The channel adopts a symmetrical hourglass shape with wide aqueous vestibules on both sides of the membrane (Figure 5d).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisecitation

    Both R22 and the Na+ are essential for fluoride permeation.

    “This Na+, together with a universally conserved arginine residue, R22, render the vestibules electropositive, and both R22 and the Na+ are essential for fluoride permeation(75).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [75] McIlwain BC, Martin K, Hayter EA, Stockbridge RB. 2020. An Interfacial Sodium Ion is an Essential Structural Feature of Fluc Family Fluoride Channels. J Mol Biol PMC7054162 31945374 source

  • It may state two separate claims as one.

    Premisecitation

    The structural Na+ ion is deeply buried, stably bound, and likely inserted upon dimer assembly.

    “This structural Na+ ion is deeply buried, stably bound, and likely inserted upon dimer assembly(75).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [75] McIlwain BC, Martin K, Hayter EA, Stockbridge RB. 2020. An Interfacial Sodium Ion is an Essential Structural Feature of Fluc Family Fluoride Channels. J Mol Biol PMC7054162 31945374 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds. It may state two separate claims as one.

    Interpretationnone given

    The Flucs are optimized for fluoride selectivity and rapid conduction, all accomplished without any depletion of the microbe's energy stores.

    “Whatever the biophysical basis, the Flucs are impeccably optimized for fluoride selectivity and rapid conduction, all accomplished without any depletion of the microbe’s energy stores.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    Many other diverse membrane protein folds share this construction, which presumably arose from the duplication and fusion of a primal homodimer.

    “Many other diverse membrane protein folds share this construction, which presumably arose from the duplication and fusion of a primal homodimer (69, 70).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [69] Forrest LR. 2015. Structural Symmetry in Membrane Proteins. Annu Rev Biophys 44: 311– 37 26098517 PMC5500171 source
    • cites [70] Keller R, Ziegler C, Schneider D. 2014. When two turn into one: evolution of membrane transporters from half modules. Biol Chem 395: 1379– 88 25296672 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    Because F− requires the smallest number of coordinating ligands of any anion, it is plausible that only by adding ligands to the fluoride binding sites could the channel be engineered to bind other anions.

    “Because F− requires the smallest number of coordinating ligands of any anion, it is plausible that only by adding ligands to the fluoride binding sites could the channel be engineered to bind other anions.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Fluoride requires the smallest number of coordinating ligands of any anion.

  • It may state two separate claims as one.

    Interpretationreasoning

    Some evidence suggests that E88 contributes to anion selectivity, although its exact role is unclear.

    “Some evidence suggests that E88 contributes to anion selectivity, although its exact role is unclear.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premiseown data

    Although the identity of polar track residues is not conserved among Fluc proteins, the polar, hydrogen-bond donating nature is conserved.

    “Sequences alignments show that although the identity of the polar track residues is not conserved among Fluc proteins, the polar, hydrogen-bond donating nature is (Figure 6c).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisereasoning

    N43 is situated between F1 and F2, with the sidechain amide coordinating F2.

    “N43 is situated between F1 and F2, with the sidechain amide coordinating F2.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Premisenone given

    The polar track is defined by a hydrogen bond donor at every fourth position, or one per helical turn.

    “Termed the polar track, this stretch of sequence is defined by a hydrogen bond donor at every fourth position, or one per helical turn (indicated with asterisks in Figure 6a).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premiseown data

    F82 and F85 are arranged adjacent to the polar track in a symmetrical box configuration, with the electropositive edge of each Phe adjacent to the electronegative face of the next Phe.

    “These highly conserved sidechains are arranged adjacent to the polar track in a symmetrical box configuration, with the electropositive edge of each Phe adjacent to the electronegative face of the next Phe.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Interpretationreasoning

    The Fluc family has not evolved into a diversity of physiological roles like the CLCs; the Flucs are very specialized in what they do.

    “It seems significant that the Fluc family has not evolved into a diversity of physiological roles like the CLCs: the Flucs are very specialized in what they do.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    The resultant protomers assemble as obligate antiparallel heterodimers where both subunits are required for channel assembly and function.

    “the resultant protomers assemble as obligate antiparallel heterodimers where both subunits are required for channel assembly and function(2).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Stockbridge RB, Robertson JL, Kolmakova-Partensky L, Miller C. 2013. A family of fluoride-specific ion channels with dual-topology architecture. Elife 2: e01084 23991286 PMC3755343 source

  • It may state two separate claims as one.

    Premisecitation

    E88 is extremely well-conserved among fluoride channels and follows the same pattern of conservation/degradation in the different domains of the eukaryotic channels as other pore-lining residues.

    “This glutamate is extremely well-conserved among fluoride channels and follows the same pattern of conservation/degradation in the different domains of the eukaryotic channels as other pore-lining residues(73).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [73] Stockbridge RB, Kolmakova-Partensky L, Shane T, Koide A, Koide S, 2015. Crystal structures of a double-barrelled fluoride ion channel. Nature 525: 548– 51 26344196 PMC4876929 source

  • It may state two separate claims as one.

    Premisenone given

    The two 15-kDa subunits in the dimer, each composed of four transmembrane helices, adopt identical structures, such that the channels possess two-fold symmetry about an axis parallel to the plane of the membrane.

    “The two 15-kDa subunits in the dimer, each composed of four transmembrane helices, adopt identical structures, such that the channels possess two-fold symmetry about an axis parallel to the plane of the membrane (Figure 5b).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premiseown data

    The substituted methionine in the F82/F85 mutant adopts a twisted conformation, filling approximately the same space as the Phe ring in wildtype, with the electropositive g-methylene sitting in the same position as the Phe edge.

    “The structure of this mutant shows that the substituted methionine adopts a twisted conformation, filling approximately the same space as the Phe ring in the wildtype protein, with the electropositive g-methylene sitting in the same position as the Phe edge (Figure 6d).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisereasoning

    CLCFs provide 5-6 ligands to each anion binding site and are less selective than Fluc.

    “This sparsity of ligands in Fluc draws a contrast with the 5–6 ligands provided to each anion binding site by the less-selective CLCFs.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    Such a dual topology architecture is exceedingly rare among membrane proteins, and has only been structurally confirmed for two other transporter classes.

    “Such an architecture is exceedingly rare among membrane proteins, and has only been structurally confirmed for two other transporter classes (71, 72).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [71] Ubarretxena-Belandia I, Baldwin JM, Schuldiner S, Tate CG. 2003. Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer. EMBO J 22: 6175– 81 14633977 PMC291852 source
    • cites [72] Assur Sanghai Z, Liu Q, Clarke OB, Belcher-Dufrisne M, Wiriyasermkul P, 2018. Structure-based analysis of CysZ-mediated cellular uptake of sulfate. Elife 7 PMC5967866 29792261 source

  • It may state two separate claims as one.

    Premisecitation

    Bacterial Flucs assemble as dual topology dimers, with one subunit inserted into the membrane with the termini facing out, and the other inserted with the termini facing in.

    “Remarkably, the bacterial Flucs embody this evolutionary antecedent, assembling as dual topology dimers, with one subunit inserted into the membrane with the termini facing out, and the other inserted into the membrane with the termini facing in (2, 60).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [2] Stockbridge RB, Robertson JL, Kolmakova-Partensky L, Miller C. 2013. A family of fluoride-specific ion channels with dual-topology architecture. Elife 2: e01084 23991286 PMC3755343 source
    • cites [60] Stockbridge RB, Koide A, Miller C, Koide S. 2014. Proof of dual-topology architecture of Fluc F- channels with monobody blockers. Nat Commun 5: 5120 25290819 PMC4265568 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.

    Premisenone given

    Hydroxyl sidechains are prevalent as hydrogen bond donors for fluoride in CLCF and Fluc proteins.

    “Some of these are expected, like the prevalence of hydroxyl sidechains as hydrogen bond donors for fluoride.”
    Quote in the paperNot a substantial claim
65 of 65

  • The paper relies on this without support.

    The proton motive force is maintained by metabolizing cells.

    “An extra thermodynamic push is provided by the proton motive force that metabolizing cells maintain.”

  • The paper relies on this without support.

    Ionic throughput is a critical functional feature of transport proteins.

    “since ionic throughput is a critical functional feature of transport proteins.”

  • The paper relies on this without support.

    F− ions are dehydrated for transport.

    “Because the F− ions are dehydrated for transport, this energetic barrier must be surmounted.”

  • The paper relies on this without support.

    Fluoride is a stronger base than the other halides and pseudohalides, and its conjugate acid HF is a weaker acid.

    “Another unique aspect of fluoride is that it is a stronger base than the other halides and pseudohalides (and its conjugate acid, HF, a weaker acid).”

  • The paper relies on this without support.

    Anions such as Cl− and NO3− are exclusively ionized in aqueous media.

    “Whereas anions such as Cl− and NO3− are exclusively ionized in aqueous media”

  • The paper relies on this without support.

    All members of the CLC family possess the gating glutamate.

    “All members of the CLC family possess this so-called gating glutamate.”

  • The paper relies on this without support.

    Compared with other CLC transporters, the aqueous vestibules of CLCF are deeper and the anhydrous zone crossed by the permeant ions is shorter.

    “Compared with other CLC transporters, the aqueous vestibules of CLCF are deeper and the anhydrous zone crossed by the permeant ions is shorter.”

  • The paper relies on this without support.

    The second family of membrane proteins associated with the fluoride riboswitches is entirely dedicated to fluoride transport.

    “The second family of membrane proteins associated with the fluoride riboswitches is entirely dedicated to fluoride transport, and its members are unlike any previously characterized protein.”

  • The paper relies on this without support.

    Members of the second family of membrane proteins associated with the fluoride riboswitches are unlike any previously characterized protein.

    “and its members are unlike any previously characterized protein.”

  • The paper relies on this without support.

    Genes encoding these proteins, originally called crcB, are found in bacteria, archaea, unicellular eukaryotes, fungi, plants, and filter feeding ocean animals like sponges and sea anemones.

    “Genes encoding these proteins, originally called crcB, are found not only in bacteria, but also in archaea, unicellular eukaryotes, fungi, plants, and filter feeding ocean animals like sponges and sea anemones.”

  • The paper relies on this without support.

    Fluc channels are constitutively open.

    “Unlike many gated channels, which open only in response to specific stimuli, the Fluc channels are constitutively open.”

  • The paper relies on this without support.

    The Fluc fold is unique among known membrane proteins.

    “The Fluc fold is unique among known membrane proteins.”

  • The paper relies on this without support.

    There is no known precedent among membrane proteins for such a non-transported structural Na+.

    “Although there is no known precedent among membrane proteins for such a non-transported structural Na+,”

  • The paper relies on this without support.

    Fluoride requires the smallest number of coordinating ligands of any anion.

    “Because F− requires the smallest number of coordinating ligands of any anion, it is plausible that only by adding ligands to the fluoride binding sites could the channel be engineered to bind other anions.”

  • The authors state this as an assumption.

    Sidechain rearrangement is limited.

    “The proposed fluoride binding sites in Fluc may be “under-coordinated,” with few protein ligands (three or four, assuming limited sidechain rearrangement) in order to exclude Cl−.”
15 of 15

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • One mechanism through which Pseudomonads handle fluoride toxicity is sensing and export of fluoride out of the cell through the CLCF family of F-/H+ antiporters and/or the Fluc/FEX family of F- channels.

    “One mechanism through which Pseudomonads handle fluoride toxicity is the sensing and export of fluoride out of the cell through the CLCF family of F−/H+ antiporters and/or the Fluc/FEX family of F− channels (McIlwain et al., 2021; Wackett, 2022a).”

    From A review of microbial degradation of per- and polyfluoroalkyl substances (PFAS): Biotransformation routes and enzymes (2023) · cites this paper as McIlwain et al., 2021

    The ‘Biological response to F− toxicity’ section describes fluoride-binding riboswitches associated with CLCF and Fluc exporter genes, and the Introduction identifies CLCFs as F−/H+ antiporters and Fluc/FEX proteins as fluoride channels. These passages support the general sensing-and-export mechanism in microbes, but do not establish that Pseudomonads specifically use it. The paper also notes that some fluoride exporters are expressed without a riboswitch. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • CrcB is known to be involved in F− detoxification in several bacterial species.

    “given the known involvement of CrcB in F− detoxification in several bacterial species (McIlwain et al., 2021)”

    From Role of the CrcB transporter of Pseudomonas putida in the multi‐level stress response elicited by mineral fluoride (2022) · cites this paper as McIlwain et al., 2021

  • Fluoride inhibits essential enzymes in central metabolism, often by forming tight complexes with metallo-cofactors like calcium or magnesium within the enzyme's active site.

    “Unlike other monovalent anions like chloride, fluoride inhibits essential enzymes in central metabolism, often by forming tight complexes with metallo-cofactors like calcium or magnesium within the enzyme’s active site65.”

    From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [65]

  • Microorganisms have evolved a suite of fluoride detoxification mechanisms.

    “Microorganisms have contended with environmental mineral fluoride over evolutionary time, evolving a suite of detoxification mechanisms.”

    From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [65]

  • Fluoride produced intracellularly and released, or produced in the periplasm, readily re-enters and accumulates in cells via weak acid accumulation.

    “Moreover, fluoride produced intracellularly and released, or produced in the periplasm, readily re-enters and accumulates in cells via a process called weak acid accumulation65,71, especially as the medium pH acidifies, which is typical with dense bacterial cultures.”

    From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [65]

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 4

  1. U.S. Dep, Public Health Rep, 130, 318
  2. CDC (Cent. Dis. Control Prev.), CDC Div. Oral Health
  3. Adamek E, 2005, Ann. Acad. Med. Stetin., 51, 69
  4. Lellouche J, 2012, Int. J. Nanomed., 7, 5611

Cited by

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Related

Metadata

Authors
McIlwain, B. C., Ruprecht, M. T., & Stockbridge, R. B.
Affiliations
University of Michigan (US)
Corresponding author
Stockbridge, Randy B.
ORCID
Venue
Annual Review of Biochemistry
Publisher
Annual Reviews
Published
2021-06-20
Volume
90
Issue
1
Pages
559-579
ISSN
  • 0066-4154 (ISSN-L)
  • 1545-4509
DOI
10.1146/annurev-biochem-071520-112507
PMC
PMC8217344
OpenAlex
W3124501889
Semantic Scholar
3f169fb6a4184b59398936b2a750753e5f41e7c8
Cited by
69
References
101
Citations by year
2026: 13 · 2025: 20 · 2024: 17 · 2023: 9 · 2022: 6 · 2021: 4
FWCI
0.94
Type
journal article
Language
en
Open access
Locations
  • Annual Review of Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health
  • National Institute of General Medical Sciences (R35GM128768)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › ATP Synthase and ATPases Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion Transport and Channel Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion channel regulation and function
Keywords
fluoride, membrane transporters, xenobiotic detoxification
MeSH terms
Cytoplasm, Fluorides, Ion Channels, Membrane Proteins, Protein Conformation, Ion Transport, Antiporters, Chloride Channels, Saccharomyces cerevisiae Proteins

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