A family of fluoride-specific ion channels with dual-topology architecture

The 'Fluc' family of small membrane proteins comprises the CrcB F− channel of Escherichia coli.

“a group of small membrane proteins generically known as the ‘Fluc’ family, which comprises the CrcB F− channel of Escherichia coli (Last et al., 2016; Stockbridge et al., 2013)”

CrcB export proteins function as passive fluoride-specific ion channels that require Na+ ions for transport activity.

“In contrast, CrcB export proteins function as passive fluoride-specific ion channels that require Na+ ions for transport activity (4, 5, 23–26).”

Both subunits must be present for a functional heterodimer Fluc F− channel.

“Given that both subunits must be present for a functional heterodimer Fluc F− channel (27)”

A. bakii has a Fluc family F− channel, a heterodimer composed of two distinct proteins encoded by crcB1 and crcB2 genes.

“A. bakii has a Fluc family F− channel, which is a heterodimer composed of two distinct proteins encoded by crcB1 and crcB2 genes (26, 27).”

Oral eubiota including S. gordonii, S. oralis, and S. sanguinis export F- via Fluc proteins.

“By contrast, oral eubiota including S. gordonii, S. oralis, and S. sanguinis all export F- via the action of Fluc (Fluoride channel) proteins (also known by their gene name, crcB), which harness the electrical component of the proton motive force to expel cytoplasmic fluoride (23).”

Streptococcus gordonii exports fluoride using a Fluc fluoride channel.

“oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel.”

Single Fluc channels can be monitored in planar lipid bilayer electrophysiological recordings, where fluoride conductances correspond to a throughput of ~10^5-6 ions/sec.

“In harmony with an electrodiffusive mechanism, single Fluc channels can be monitored in planar lipid bilayer electrophysiological recordings, where the fluoride conductances observed correspond to a throughput of ~105–6 ions/sec(2, 61).”

Proteins encoded by the crcB genes are electrodiffusive F− channels.

“Electrophysiological characterization showed that the proteins encoded by the crcB genes are electrodiffusive F− channels (2, 3, 60–63), and they were renamed Fluc (Fluoride channel) in bacteria and FEX (Fluoride exporter) in eukaryotes.”

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).”

Flucs assemble as unusual antiparallel dimers, with one protomer facing out and the other facing in.

“The Flucs assemble as unusual antiparallel dimers, where one protomer is inserted into the membrane facing out, and the other is inserted into the membrane facing in81,83.”

Flucs function as passive fluoride channels that exploit the positive-outside membrane potential to drive expulsion of fluoride.

“In contrast to the CLCFs, the Flucs (Fluoride channel) function as passive fluoride channels81 that exploit the positive-outside membrane potential (the electrical component of the proton-motive force) to drive the expulsion of this anionic species71.”

Flucs have a rapid rate of F− efflux (10^6 ions/s) and extremely high selectivity for fluoride over other anions, exceeding 10,000-fold.

“The Flucs are notable for their rapid rate of F− efflux (106 ions/s) and extremely high selectivity for fluoride over other anions, which exceeds 10,000-fold81,82.”

The most common gene associated with fluoride riboswitches, crcB (also called fex or fluc), codes for a fluoride-specific channel protein.

“The most common gene associated with fluoride riboswitches, termed crcB (also called fex, or fluc), codes for a fluoride-specific channel protein (Baker et al., 2012; Li et al., 2013; Stockbridge et al., 2013).”

Fluc-type ion channels permit the selective and passive diffusion of fluoride through membranes.

“Curiously, evidence presented to date convincingly demonstrates that Fluc-type ion channels permit the selective and passive diffusion of fluoride through membranes (Stockbridge et al., 2013; Ji et al., 2014)”

The large negative membrane potential of bacterial cells should greatly favor fluoride efflux through the channel.

“However, the large negative membrane potential of bacterial cells should greatly favor fluoride efflux through the channel (Stockbridge et al., 2013).”

Under slightly acidic conditions, fluoride is expected to accumulate in cells relative to the environment, and therefore fluoride outflow should again be favored.

“Also, under slightly acidic conditions, fluoride is expected to accumulate in cells relative to the environment (Stockbridge et al., 2013; Li et al., 2013), and therefore fluoride outflow should again be favored.”

Fluc channels exhibit extreme selectivity for F- over Cl- and dual-topology dimeric assembly.

“including extreme selectivity for F- over Cl- and dual-topology dimeric assembly5–6.”

Two-pore assembly neatly accounts for evolutionary drift in eukaryotic Fluc channels, all of which consist of an inverted repeat of two homologous Fluc domains fused into a single polypeptide.

“Two-pore assembly neatly accounts for evolutionary drift in eukaryotic Fluc channels, all of which consist of an inverted repeat of two homologous Fluc domains fused into a single polypeptide4,5.”

Two-pore behavior is not apparent in single-channel recordings since both pores are nearly always open.

“Two-pore behavior is not apparent in single-channel recordings since both are nearly always open5.”

Fluc proteins are ion channels with high specificity (>10^4) for F- over Cl- and a dual-topology dimeric architecture.

“We recently established5,6 that Fluc proteins are ion channels with two unusual properties: an exceedingly high specificity (>104) for F- over Cl–, and a dual-topology dimeric architecture”

The double-pore assembly idea does not clash with any electrophysiological experiments, and double-pore assembly was explicitly cited previously as a possible though unlikely architecture consistent with the channel's functional behaviour.

“Though unexpected, this idea does not clash with any electrophysiological experiments, and double-pore assembly was explicitly cited previously as a possible though unlikely architecture consistent with the channel’s functional behaviour5.”

Nearly all microorganisms possess membrane proteins dedicated to the export of fluoride ion (F−).

“Nearly all microorganisms possess membrane proteins dedicated to the export of fluoride ion (F−).”

The Fluc/FEX family are dedicated fluoride channels that permit electrodiffusive export of F−.

“The second family, called Fluc (Fluoride channel) in bacteria and FEX (Fluoride exporter) in eukaryotes, are dedicated fluoride channels that permit electrodiffusive export of F− (2, 3).”

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).”

Fluoride export proteins belong to one of two families: the CLCF family and the Fluc/FEX family.

“These proteins belong to one of two families, each with a completely different fold, and that export F− via fundamentally different mechanisms.”

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).”

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).”