This project will subject two newly discovered families of F- -transporting membrane proteins to detailed functional and mechanistic scrutiny and will seek to solve their high-resolution crystal structures. These proteins expel F- from the cytoplasm to protect bacteria and unicellular eukaryotes from the toxic effects of ambient F- in the environment. The two protein families are phylogenetically unrelated. The """"""""CLCF"""""""" exporters represent a clade within the long-studied CLC superfamily of anion channels and transporters, while the """"""""Fluc"""""""" exporters are a previously unknown-function family of small membrane proteins. Our preliminary experiments have already uncovered for the CLCF exporters several surprising variations on mechanistic themes well-established for Cl- transport in """"""""conventional"""""""" CLC proteins: (1) the absence of the anion- coordinating residues conserved among all previously studied CLCs, (2) an extremely high selectivity for F-, (3) a proton-coupled F- antiport mechanism despite a signature sequence suggesting that these would be ion channels, and (4) an unprecedented 1-to-1 anion/H+ exchange stoichiometry. For Fluc proteins, our work shows these to be highly F--selective ion channels. Sequence analysis argues strongly that the functional channel is an unusual """"""""antiparallel oligomer,"""""""" and our experimental results indicate unprecedented dimeric architecture in which the twin subunits are inserted into the membrane in opposite orientations. The project combines electrophysiological, membrane-biophysical, and structural analysis to attack fundamental questions arising from these results: what residues determine anion- selectivity and H+ movement in the CLCF antiporters? How must we modify accepted antiport mechanisms to account for the surprising 1-to-1 F-/H+ stoichiometry of CLCFs? Where are the pore-lining residues in Fluc channels and what accounts for their high anion selectivity? Answers to basic questions like these are necessary to bring into focus our view of how these membrane proteins work to export F- and thus counteract this ion's pervasive challenge to cellular integrity. Since these F- exporters are found in many bacterial and eukaryotic pathogens but not in vertebrates, they may provide novel antibiotic targets.

Public Health Relevance

This project is aimed at fundamental properties of a very new and unusual class of membrane proteins, and as such falls into the category of basic research. However, the fluoride exporters under study here protect many pathogenic organisms from environmental fluoride (e.g., Mycobacterium tuberculosis, Enterococcus caselliflavus, Candida albicans, Leishmania major, Toxoplasma gondii), but are not found in any vertebrate genomes. These might therefore eventually provide unique targets for novel antibiotics, since environmental F- is forever all around us and must be dealt with by unicellular microorganisms.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
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Brandeis University
Schools of Arts and Sciences
United States
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Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew et al. (2016) Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex. Elife 5:
Last, Nicholas B; Kolmakova-Partensky, Ludmila; Shane, Tania et al. (2016) Mechanistic signs of double-barreled structure in a fluoride ion channel. Elife 5:
Stockbridge, Randy B; Kolmakova-Partensky, Ludmila; Shane, Tania et al. (2015) Crystal structures of a double-barrelled fluoride ion channel. Nature 525:548-51
Turman, Daniel L; Nathanson, Jacob T; Stockbridge, Randy B et al. (2015) Two-sided block of a dual-topology F- channel. Proc Natl Acad Sci U S A 112:5697-701
Last, Nicholas B; Miller, Christopher (2015) Functional Monomerization of a ClC-Type Fluoride Transporter. J Mol Biol 427:3607-3612
Ji, Chunhui; Stockbridge, Randy B; Miller, Christopher (2014) Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. J Gen Physiol 144:257-61
Brammer, Ashley E; Stockbridge, Randy B; Miller, Christopher (2014) F-/Cl- selectivity in CLCF-type F-/H+ antiporters. J Gen Physiol 144:129-36
Stockbridge, Randy B; Koide, Akiko; Miller, Christopher et al. (2014) Proof of dual-topology architecture of Fluc F- channels with monobody blockers. Nat Commun 5:5120