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

Abstract

Fluoride ion, ubiquitous in soil, water, and marine environments, is a chronic threat to microorganisms. Many prokaryotes, archea, unicellular eukaryotes, and plants use a recently discovered family of F− exporter proteins to lower cytoplasmic F− levels to counteract the anion’s toxicity. We show here 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. This dual-topology assembly has not previously been seen in ion channels but is known in multidrug transporters of the SMR family, and is suggestive of an evolutionary antecedent of the inverted repeats found within the subunits of many membrane transport proteins.DOI: http://dx.doi.org/10.7554/eLife.01084.001

Two-sided block of a dual-topology F − channel

“…1B illustrate block of the bacterial Fluc homolog Bpe by the L3 monobody, a previously described channel-monobody pair (2,3). In the absence of blocker, Bpe adopts a conducting state with open probability greater than 99%; infrequent closings of millisecond durations are filtered out in the recordings presented here, and in all figures, channel traces are displayed opening upward, regardless of voltage polarity.…”

“…Phospholipids (1-palmitoyl, 2-oleoyl phosphaditylethanolamine or the analogous phosphatidyl glycerol) were obtained from Avanti Polar Lipids, fluorophore-maleimide was from Molecular Probes, and n-decylmaltoside was from Anatrace. Amino acid sequence, expression, purification, and reconstitution of the Fluc homolog used here, which we denote Bpe, and monobody L3 were as described (2,3). The Bpe construct used here carries two functionally nonperturbing mutations (R29K/E94S) to enhance biochemical tractability.…”

“…The novelty of this previously unsuspected microbial physiology is mirrored in the unusual molecular architecture of one class of these exporters, the Fluc family. Flucs are highly F − -selective ion channels built as dual-topology homodimers, wherein the paired subunits of the functional channel assemble in antiparallel transmembrane topology (2,3). This architecture demands that, if the twin subunits adopt identical conformations, the channel must present structurally identical ion entryways to the two sides of the membrane, in sharp contrast to the parallel assembly of conventional multisubunit membrane proteins.…”

Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains

“…CLC F genes encode F Ϫ /H ϩ antiporters in eubacteria and are generally under the control of F Ϫ -sensitive riboswitches (8,14). The second, much more broadly distributed family is called Fluc in bacteria (formerly crcB) (15) and FEX 2 in eukaryotes (16). Several Fluc proteins have been demonstrated to function as bona fide ion channels that can discriminate against the chloride ion with Ͼ10,000-fold selectivity (15).…”

“…The second, much more broadly distributed family is called Fluc in bacteria (formerly crcB) (15) and FEX 2 in eukaryotes (16). Several Fluc proteins have been demonstrated to function as bona fide ion channels that can discriminate against the chloride ion with Ͼ10,000-fold selectivity (15). Among eukaryotic systems, Saccharomyces, Candida, and Neurospora have been shown to depend upon FEX proteins for resistance to fluoride (16), but neither the mode of transport nor the anion selectivity of those proteins has been established.…”