1.A.43 The Camphor Resistance or Fluoride Channel (Fluc) Family

CrcB of E. coli appears to confer camphor resistance when overexpressed and induces resA expression (Sand et al. 2003). Deletion is not lethal but increases sensitivity to camphor and fluoride. CrcB has 127 aas and 4 TMSs. Its many close homologues are small proteins, usually with 4 TMSs.  These proteins mediate fluoride resistance (Ji et al. 2014).  The active transporter is a dimer of 4 TMS subunits arranged antiparallel (Stockbridge et al. 2014).

Fluoride, F-, ubiquitous in soil, water and marine environments, is a chronic threat to microorganisms. Many bacteria, archaea, unicellular eukaryotes and plants use F- exporters to lower cytoplasmic F- levels to counteract the anion's toxicity. Stockbridge et al. 2013 and Li et al. 2013 showed that these ''Fluc'' proteins, purified and reconstituted in liposomes and planar phospholipid bilayers, form constitutively open anion channels with extreme selectivity for F- over Cl-. The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation, or a single protein with two internal 4TMS repeats (as for the yeast proteins (TC# 1.A.43.2.4 and 5). This dual-topological assembly had not previously been seen in ion channels but is known in multidrug transporters of the SMR sub-family of the DMT superfamily (TC# 2.A.7.1) (Pornillos et al. 2005; Pornillos and Chang 2006). 

Crystal structures showed that Fluc channels contain two separate ion-conduction pathways, each with two F- binding sites (Stockbridge et al. 2015). Last et al. 2016 examined the consequences of mutating two conserved F--coordinating phenylalanine residues. Substitution of each phenylalanine specifically extinguished its associated F- binding site in crystal structures and concomitantly inhibited F- permeation. Functional analysis of concatemeric channels, which permit mutagenic manipulation of individual pores, showed that each pore can be separately inactivated without blocking F- conduction through its symmetry-related twin. The results strongly supported a dual-pathway architecture of these channels (Last et al. 2016).



This family belongs to the .

 

References:

Hu, K.H., E. Liu, K. Dean, M. Gingras, W. DeGraff, and N.J. Trun. (1996). Overproduction of three genes leads to camphor resistance and chromosome condensation in Escherichia coli. Genetics 143: 1521-1532.

Ji, C., R.B. Stockbridge, and C. Miller. (2014). Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. J Gen Physiol 144: 257-261.

Last, N.B., L. Kolmakova-Partensky, T. Shane, and C. Miller. (2016). Mechanistic signs of double-barreled structure in a fluoride ion channel. Elife 5:.

Li, S., K.D. Smith, J.H. Davis, P.B. Gordon, R.R. Breaker, and S.A. Strobel. (2013). Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins. Proc. Natl. Acad. Sci. USA 110: 19018-19023.

Picoli, C., E. Soleilhac, A. Journet, C. Barette, M. Comte, C. Giaume, F. Mouthon, M.O. Fauvarque, and M. Charvériat. (2019). High-Content Screening Identifies New Inhibitors of Connexin 43 Gap Junctions. Assay Drug Dev Technol 17: 240-248.

Pornillos, O. and G. Chang. (2006). Inverted repeat domains in membrane proteins. FEBS Lett. 580: 358-362.

Pornillos, O., Y.J. Chen, A.P. Chen, and G. Chang. (2005). X-ray structure of the EmrE multidrug transporter in complex with a substrate. Science 310: 1950-1953.

Sand, O., M. Gingras, N. Beck, C. Hall, and N. Trun. (2003). Phenotypic characterization of overexpression or deletion of the Escherichia coli crcA, cspE and crcB genes. Microbiology 149: 2107-2117.

Smith, K.D., P.B. Gordon, A. Rivetta, K.E. Allen, T. Berbasova, C. Slayman, and S.A. Strobel. (2015). Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains. J. Biol. Chem. 290: 19874-19887.

Stockbridge, R.B., A. Koide, C. Miller, and S. Koide. (2014). Proof of dual-topology architecture of Fluc F(-) channels with monobody blockers. Nat Commun 5: 5120.

Stockbridge, R.B., J.L. Robertson, L. Kolmakova-Partensky, and C. Miller. (2013). A family of fluoride-specific ion channels with dual-topology architecture. Elife 2: e01084.

Stockbridge, R.B., L. Kolmakova-Partensky, T. Shane, A. Koide, S. Koide, C. Miller, and S. Newstead. (2015). Crystal structures of a double-barrelled fluoride ion channel. Nature 525: 548-551.

Examples:

TC#NameOrganismal TypeExample
1.A.43.1.1

The camphor resistance protein, CrcB or FluC (Hu et al. 1996; Sand et al. 2003).  Exports fluoride selectively over chloride by an anion open channel mechanism (Stockbridge et al. 2013).  The active transporter is a dimer of 4 TMS subunits arranged in an antiparallel transmembrane orientation (Stockbridge et al. 2014).  In bacteria lacking Fluc, F- accumulates when the external medium is acidified as a predicted function of the transmembrane pH gradient. This weak acid accumulation effect, which results from the high pKa (3.4) and membrane permeability of HF, is abolished by Fluc channels (Ji et al. 2014).  A proper tubulin network is required for functional Cx43 GJ channels, and mefloquineis a gap junction inhibitor (Picoli et al. 2019).

.

Bacteria

CrcB of E. coli (P37002)

 
1.A.43.1.10

CrcB-like protein of 164 aas and 4 TMSs

Actinobacteria

CreB of Mobiluncus curtisii (Falcivibrio vaginalis)

 
1.A.43.1.11

Putative fluoride transporter of 122 aas, CrcB

Proteobacteria

CrcB of Campylobacter jejuni

 
1.A.43.1.12

CreB of 168 aas and 4 TMSs

CreB of Brachybacterium faecium

 
1.A.43.1.13

CreB of 123 aas and 4 TMSs.

CreB of Aequorivita sublithincola

 
1.A.43.1.14

CrcB, putative fluoride channel protein of 124 aas and 4 TMSs

CrcB of Lactobacillus kefiranofaciens

 
1.A.43.1.15

CrcB of 133 aas and 4 TMSs.

CrcB of Halorubrum coriense

 
1.A.43.1.16

Fluc homologue of 453 aas and 9 putative TMSs.

Fluc of Acanthamoeba castellanii

 
1.A.43.1.17

Fluoride ion channel of 128 aas and 4 TMSs, Fluc or CrcB.  The crystal structure is known (PDB5A40; 5A43).

Fluc of Bordetella pertussis

 
1.A.43.1.2

Protein CrcB homologue 2

Bacilli

crcB2 of Bacillus subtilis

 
1.A.43.1.3

Putative fluoride-selective channel of 143 aas and 4 TMSs, CrcB.

Actinobacteria

CreB of Propionibacterium acnes

 
1.A.43.1.4

CreB homologue of 124 aas

Euryarchaea

CrcB homologue of Methanocaldococcus fervens

 
1.A.43.1.5

CrcB homologue of 172 aas and 4 TMSs

Proteobacteria

CrcB of Parvularcula bermudensis

 
1.A.43.1.6

Putative fluoride exporter, CrcB.

Chlorobi

CrcB of Pelodictyon luteolum (Chlorobium luteolum)

 
1.A.43.1.7

Putative fluoride exporter, CrcB if 114 aas and 4 TMSs.

Euryarchaea

CrcB of Thermococcus barophilus

 
1.A.43.1.8

Uncharacterized protein of 151 aas and 4 TMSs.

Actinobacteria

UP of Rothia mucilaginosa (Stomatococcus mucilaginosus)

 
1.A.43.1.9

Putative fluoride exporter, CrcB of 113 aas and 4 TMSs.

Euryarchaea

UP of Haloquadratum walsbyi

 
Examples:

TC#NameOrganismal TypeExample
1.A.43.2.1

CrcB-like protein of 307 aas and 6 TMSs in an apparent 3 + 3 arrangement.

Ciliates

CrcB homologue of Tetrahymena thermophila

 
1.A.43.2.2

Uncharacterized protein of 372 aas and 9 - 10 TMSs

Fungi

UP of Kazachstania africana (Kluyveromyces africanus)

 
1.A.43.2.3

CrcB domain containing protein of 310 aas and 9 TMSs in a 4 + 5 arrangement, with both halves showing sequence similarity with the 4 TMS CrcB of E. coli.

Yeast

CrcB homologue of Schizosaccharomyces cryophilus

 
1.A.43.2.4

Plasma membrane fluoride ( > chloride) export channel of 375 aas and 8 TMSs, FEX1 (Li et al. 2013). The two homologous 4 TMS domains are functionally assymetric (Smith et al. 2015).  There are two very similar fex genes in S. cerevisiae, the other having TC# 1.A.43.2.5. Fex1 is consitutively synthesized (Smith et al. 2015).

Fungi

FEX1 of Saccharomyces cerevisiae

 
1.A.43.2.5

Fluoride exporter, FEX2 of 375 aas and 8 TMSs (Li et al. 2013).

Fungi

FEX2 of Saccharomyces cerevisiae

 
1.A.43.2.6

Fluoride exporter, FEX, of 526 aas and 10 putative TMSs (Li et al. 2013).

Fungi

FEX of Neurospora crassa

 
1.A.43.2.7

Camphor resistance CrcB protein of 461 aas and 9 putative TMSs.

Plants

CrcB of Arabidopsis thaliana

 
1.A.43.2.8

Uncharacterized protein of 405 aas and 8 putative TMSs.

Animals

UP of Ciona intestinalis (Transparent sea squirt) (Ascidia intestinalis)

 
1.A.43.2.9

Uncharacterized protein of 460 aas and 9 TMSs/

UP of Phytophthora parasitica

 
Examples:

TC#NameOrganismal TypeExample
1.A.43.3.1

CreB of 346 aas and 4 TMSs.

CreB of Bifidobacterium longum

 
1.A.43.3.2

Putative fluoride channel, CrcB, of 180 aas and 4 TMSs.

CrcB of Scardovia wiggsiae

 
1.A.43.3.3

Putative fluoride ion channnel, CrcB, of 178 aas and 4 TMSs

CrcB of Bifidobacterium longum

 
1.A.43.3.4

Putative fluoride ion channel, CrcB, with 310 aas and 4 TMSs.

CrcB of Bifidobacterium animalis subsp. lactis