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The Formate/Nitrite Transporter Family Of Anion Channels.

Article  in  Biological Chemistry · February 2013

DOI: 10.1515/hsz-2012-0339 · Source: PubMed

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 DOI 10.1515/hsz-2012-0339      Biol. Chem. 2013; 394(6): 715–727

Review

Wei Lü, Juan Du, Nikola J. Schwarzer, Tobias Wacker, Susana L.A. Andrade and Oliver Einsle*

The formate/nitrite transporter family of anion

channels
Abstract: The formate/nitrite transporter (FNT) family of Introduction
integral membrane proteins comprises pentameric chan-
nels for monovalent anions that exhibit a broad specificity
Biological membranes are highly impermeable for polar
for small anions such as chloride, the physiological cargo
or charged molecules, and the translocation of ions thus
molecules formate, nitrite, and hydrosulfide, and also
requires the action of dedicated, membrane-integral trans-
larger organic acids. Three-dimensional structures are
porters or channels (Singer and Nicolson, 1972; Singer,
available for the three known subtypes, FocA, NirC, and
1990). Such proteins face the challenging task of provid-
HSC, which reveal remarkable evolutionary optimizations
ing a high degree of selectivity for the transported species
for the respective physiological context of the channels.
that leaves other essential ion gradients unaffected and,
FNT channels share a conserved translocation pathway
at the same time, enables high transport rates. If the
in each protomer, with a central hydrophobic cavity that
action of the membrane transporter or channel results in
is separated from both sides of the membrane by a nar-
a net translocation of charge across the membrane, the
row constriction. A single protonable residue, a histidine,
type of transport is classified as electrogenic and is quan-
plays a key role by transiently protonating the transported
tified by its conductance G, commonly in units of siemens
anion to allow an uncharged species to pass the hydro-
(S) (S = A/V) (Hille, 2001). In general, cation channels are
phobic barrier. Further selectivity is reached through
highly selective, likely because the transmembrane gradi-
variations in the electrostatic surface potential of the pro-
ent of the smallest and most elusive cation, the proton, is
teins, priming the formate channel FocA for anion export,
essential for the viability of almost every living cell. Simi-
whereas NirC and HSC should work bidirectionally. Elec-
larly, functional and distinct gradients of Na+, K+, or Ca2+
trophysiological studies have shown that a broad variety
are required for the central signaling processes (Latorre
of monovalent anions can be transported, and in the case
and Miller, 1983; Tsien et  al., 1987). Meanwhile, anions
of FocA, these match exactly the products of mixed-acid
are often only shuttled to balance charges, in particular
fermentation, the predominant metabolic pathway for
in the case of Cl−, or they serve as metabolic substrates in
most enterobacterial species.
assimilatory or dissimilatory pathways (Moir and Wood,
2001). In this context, the requirement for ion selectivity is
Keywords: electrophysiology; formate channel; hydro-
less strict, and accordingly, a higher degree of promiscuity
sulfide channel; membrane proteins; nitrite channel;
is frequently observed in anion channels (Eisenman and
protein crystallography.
Horn, 1983). The movement of ionic species against their
electrochemical potential gradient is termed active trans-
port and requires the translocation to be mechanistically
*Corresponding author: Oliver Einsle, Lehrstuhl für Biochemie, coupled to an energy source such as light or ATP hydrolysis
Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, (primary active transport) or the cotransport of a second
Albertstrasse 21, D-79104 Freiburg, Germany; and BIOSS Centre for
species along its electrochemical potential gradient (sec-
Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg,
Hebelstrasse 25, D-79104 Freiburg, Germany, ondary active transport) (Kaback, 1974; Slater et al., 1974).
e-mail: einsle@biochemie.uni-freiburg.de Few cases are known where members of the same family
Wei Lü, Juan Du, Nikola J. Schwarzer, Tobias Wacker and Susana L.A. of integral membrane proteins can act as active transport-
Andrade: Lehrstuhl für Biochemie, Institut für Biochemie, Albert- ers and passive channels. A well-characterized example is
Ludwigs-Universität Freiburg, Albertstrasse 21, D-79104 Freiburg,
the ClC family of chloride channels, where a single amino
Germany
Susana L.A. Andrade: BIOSS Centre for Biological Signalling
acid substitution can transform a passive ion channel into
Studies, Albert-Ludwigs-Universität Freiburg, Hebelstrasse 25, an active H+/Cl− antiporter (Miller, 2006). However, a direct
D-79104 Freiburg, Germany switch from active to passive transport within a single,

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716      W. Lü et al.: Formate/nitrite transporter family of anion channels

functional protein that solely depends on the metabolic
context of the cell was not described in molecular detail
so far, and a mechanism of this kind was found through in
vivo studies only in a single system.
Within the classification scheme of membrane trans-
port proteins, transporter class 2.A.44 comprises the
formate/nitrite transporter (FNT) family that is widely
distributed in particular among enteric bacteria such as
Escherichia and Salmonella species (Saier et  al., 1999).
Many of these are human pathogens, making the FNT pro-
teins targets of potential pharmacological relevance. The
members of the FNT family are the formate channel FocA
(Suppmann and Sawers, 1994; Delomenie et  al., 2007)
and the formate uptake permease FdhC (White and Ferry,
1992), the nitrite channel NirC (Clegg et al., 2002), and – as
a most recent addition – a hydrosulfide channel termed
HSC, which functions in assimilatory sulfite reduction
to sulfide (Czyzewski and Wang, 2012). FNT proteins are
small, integral membrane proteins of 27–31 kDa with six
transmembrane helices and share a substantial degree of
sequence similarity. Nevertheless, they attain diverse met-
abolic roles, and the proteins themselves reflect the evolu-
tionary adaptation to different physiological contexts. The
gene for the formate channel FocA shares an operon with
the one for pyruvate:formate lyase (PflB) (Suppmann and
Sawers, 1994). PflB is the key enzyme of anaerobic mixed-
acid fermentation, a predominant energy metabolism in
enteric bacteria that thrive on high carbohydrate levels in
the low-oxygen environment of the mammalian intestine
(Figure 1A). FocA is primarily required for the export of the
formate anion, one of the products of PflB, for the oxida-
tion to CO2 by periplasmic formate dehydrogenases, but its
functionality turns out to be far more complex, as detailed
below. The nitrite channel NirC acts as an importer for
nitrite anions for cytoplasmic reduction to ammonium,
NH4+, by the siroheme-dependent nitrite reductase NirBD
(Figure 1B). Due to the toxicity of the anion, the intracellu-
lar levels of nitrite are kept very low and the primary role of
NirC, thus, is that of a passive importer, although an indi-
cation of facultative, active transport by secondary proton
translocation exists (Jia et al., 2009). The third member of
the FNT family, the hydrosulfide channel HSC, forms part
of the asr operon for assimilatory sulfite reduction and
may consequently be designated AsrD (Figure 1C). Its task
is to remove HS−, the toxic end product of the six-electron
reduction of sulfite, SO−3 , by assimilatory sulfite reductase
AsrABC, from the cytoplasm (Czyzewski and Wang, 2012).
Although structurally closely related to NirC, it functions
as a product exporter.
During recent years, high-resolution crystal structures
of FocA, NirC, and AsrD proteins have become available,
and functional studies using electrophysiology in lipid
bilayers and solid-supported membrane (SSM) systems
have complemented earlier in vivo work. In the present
review, we summarize the current state of knowledge on
this versatile class of membrane transport proteins.

Figure 1 The FNT family of integral membrane proteins in the respective operon context.
(A) The formate channel FocA is cotranscribed with pyruvate:formate lyase (PlfB) that cleaves pyruvate into acetyl CoA and formate as the
central step of mixed-acid fermentation. FocA primarily functions as an export channel for cytoplasmic formate, the product of PflB, but also
exports other anionic end products of the pathway, such as lactate and acetate. (B) NirC is a substrate importer that translocates nitrite
anions from the bacterial periplasm to the cytoplasm, where they are reduced to ammonium by the assimilatory nitrite reductase NirBD.
(C) The hydrosulfide channel HSC is the fourth gene in an operon-encoding assimilatory sulfite reductase and may therefore be designated
AsrD. It serves as an exporter for the toxic end product of sulfite reduction, the hydrosulfide anion HS−.

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 W. Lü et al.: Formate/nitrite transporter family of anion channels      717

General properties of FNT family
channels
The first direct evidence of the existence of a specific trans-
port system for formate anions was provided by Sawers and
coworkers in 1994, when variants resistant to the cytotoxic
formate analog hypophosphite were found to contain point
mutations in a heretofore undefined open reading frame
within the operon encoding the enzyme pyruvate:formate
lyase (Suppmann and Sawers, 1994). The gene was desig-
nated focA (for ‘formate channel’), and an ortholog, fdhC,
was then also found in the fdh operon-encoding formate
dehydrogenase in Methanobacterium formicium (White
and Ferry, 1992). The presence of a similar gene in the nir
operon coding for assimilatory nitrite reductase as well as
the structural similarity of formate and nitrite later led Cole
and coworkers to assume that this gene, designated nirC,
would code for a nitrite channel (Clegg et al., 2002). Cellu-
lar studies confirmed this and showed that in Escherichia
coli NirC acted in a complex interplay with the transport-
ers NarK and NarU, which likely function as nitrite/nitrate
exchangers (Jia et al., 2009). High-resolution crystal struc-
tures became available for FocA from E. coli (Wang et al.,
2009) and Vibrio cholerae (Waight et al., 2010), both crys-
tallized at high pH. A subsequent structure determination
at pH 4 showed unexpected conformational changes at the
cytoplasmic N-termini of the pentamer, but it also under-
lined that the core of the channel remains unaltered (Lü
et al., 2011). Structural information was provided next for
the hydrosulfide channel HSC (or AsrD) from Clostridium
difficile (Czyzewski and Wang, 2012), followed by a 2.4 Å
structure of NirC from Salmonella typhimurium (Lü et al.,
2012b).
The most visually striking feature of all FNT family
members the pentameric quaternary structure that results
in the channels forming disk-shaped multimers with an
approximate thickness of 48 Å and a diameter of 80 Å
(Figure 2A) (Wang et al., 2009; Falke et al., 2010). The pen-
tamer surrounds what appears to be a central pore, but
a close inspection of the obtained electron density maps
pointed toward the presence of lipid or detergent mol-
ecules in this pore, so that it is not assumed to be con-
ducting. Instead, in each protomer, six transmembrane
helices form a barrel with a left-handed tilt, surround-
ing a narrower pore that constitutes the functional anion
channel (Figure 2B). This channel invariably contains two
tight constrictions within the membrane that separate a
hydrophobic, internal chamber from both the cytoplasmic
and periplasmic space. A histidine residue located in this
vestibule, in close proximity to the periplasmic constric-
tion, is an invariant amino acid residue in all FNT family
channels and an essential element of the transport mech-
anism. Two of the transmembrane helices, TM2 and TM5,
are split within the membrane into two separate segments
that are connected by an extended loop region. These
loops are instrumental in forming the constrictions in the
transport channel, with the TM2a-TM2b loop for the cyto-
plasmic constriction and the TM5a-TM5b loop for the peri-
plasmic one (Figure 2B).
Earlier sequence analyses did not indicate a distinct
evolutionary kinship of the FNT family of ion channels
with any other class of proteins. Unexpectedly, however,
the structure of E. coli FocA (Wang et  al., 2009) then
revealed that the topology and arrangement of the six
transmembrane helices of FocA correspond strikingly
well to those of aquaporins and glyceroporins, a family

Figure 2 The architecture of FNT channels.

(A) The proteins form stable homopentamers in the cytoplasmic membrane of their hosts. S. typhimurium FocA, seen from the periplasmic
side, shows individual substrate channels in each of the protomers, whereas the central pore is nonconductive and most likely filled with
lipid molecules. (B) Stereo view of a single protomer of the nitrite channel NirC from S. typhimurium. The transmembrane helices TM2 and
TM5 are interrupted by loop regions within the membrane that are involved in forming two constrictions within the transport channel (black
dotted surface).

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718      W. Lü et al.: Formate/nitrite transporter family of anion channels
of permeases that is unrelated to FNT proteins both in
sequence and in function (Agre et  al., 2002). A struc-
tural superposition of the E. coli aquaporin AqpZ with S.
typhimurium NirC not only underlines the resemblance
but also shows the differences that form the basis for
the very divergent functional properties of both systems
(Figure 3A). Aquaporins transport uncharged (but polar)
water molecules across membranes to maintain osmotic
balance, and they face the particular challenge that
during this transport they have to reliably preclude the
passage of protons that would lead to the deterioration of
the proton motif force. This is achieved through a finely
tuned electrostatic potential field that orients two water
molecules on different sites of a single, central constric-
tion in a way to preclude the formation of a hydrogen bond
across this constriction that could provide an unwanted
proton conduit (Agre et al., 2002). In the channels of the
Figure 3 FNT channels are topological homologues of aquaporins.
(A) Stereo representation of a superposition of the protomers of
StNirC (green, PDB-ID 4FC4) and EcAqpZ (red, PDB-ID 1RC2). The
topology of the six transmembrane helices is fully retained, includ-
ing the functionally relevant interruptions in helices TM2 and TM5.
(B) A major consequence of the slight distortions in the monomer is
a change in quaternary structure, making aquaporins and glycer-
oporins functional tetramers (red), while FNT channels form stable
pentamers (green).
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FNT family, the passage of water molecules is presumably
blocked by the hydrophobic barrier formed by two narrow
constrictions and a central vestibule lined by hydrophobic
amino acid side chains.
The slightly altered overall shape of FNT channels
with respect to aquaporins and glyceroporins is of major
consequence for the quaternary structure of the channels.
Aquaporins/glyceroporins form tetramers, whereas FNT
channels are pentameric (Figure 3B). In both cases, each
protomer holds a distinct transport channel, but at least
in the case of the formate channel FocA, the structural
data point toward a possible functional relevance of the
pentameric architecture.
The nitrite channel NirC
Nitrite (NO−2 ) is the central metabolic hub of the biogeo-
chemical nitrogen cycle (Einsle and Kroneck, 2004). It is
the product of the (reversible) two-electron reduction of
nitrate, NO3−, as well as of the four-electron oxidation of
hydroxylamine, H2NOH (Martinez-Espinosa et  al., 2011).
The reduction of nitrite can either be a one-electron
process that yields NO in the pathway of denitrification or,
alternatively, a six-electron process that produces ammo-
nium, NH4+. This reaction can occur in a dissimilatory
context, catalyzed by cytochrome c nitrite reductase in
the periplasm (Einsle, 2011), or it can be directed toward
nitrite assimilation in the cytoplasm, in which case the
siroheme-dependent nitrite reductase NirBD is used
(Berks et al., 1995). The nir operon typically contains a nirC
gene that encodes an FNT channel through which the cell
is able to import the anionic substrate of the NirBD into
the cytoplasm (Peakman et al., 1990). As an assimilatory
enzyme, NirBD is active when reduced nitrogen is required
for biosynthetic purposes and cell growth. In addition, a
second role for the Nir system was discovered recently in
pathogenic S. typhimurium that infect macrophages of the
mammalian immune system and proliferate within the
cytoplasm. As a host defense mechanism, macrophages
use inducible NO synthase to convert arginine to citrul-
line, releasing NO (Xie and Nathan, 1994). Through reac-
tion with peroxide or superoxide, this radical species can
form toxic nitrite and peroxynitrite that then cause severe
damage to the intruding cells (Chakravortty and Hensel,
2003; Brett et  al., 2008). Enterobacteria express the nir
genes as a countermeasure and import nitrite or perox-
ynitrite via the FNT channel NirC to subsequently detoxify
both anions to ammonium using NirBD. Accordingly, a
ΔnirC strain of S. typhimurium showed strongly inhibited
 W. Lü et al.: Formate/nitrite transporter family of anion channels      719

intracellular proliferation in murine macrophages, con- architectural principles detailed above, with all protom-
comitant with increased levels of NO (Das et  al., 2009). ers of the pentameric channel showing identical con-
Thus, while the Nir system will rarely be required for formations within experimental error (Figure 2B). The
assimilatory purposes in the nitrogen-rich environment of transport channel within the NirC protomer starts on the
the mammalian intestine, it has taken on a second role in cytoplasmic and periplasmic sides with large diameters,
breaching host defense. then narrows down to reach two tight constrictions within
In E. coli and S. typhimurium, the gene for NirC shares the membrane that separate a central, hydrophobic vesti-
an operon of the structure nirBDCcysG with the siroheme- bule (Figures 4 and 5). The amino acid residues forming
dependent nitrite reductase NirBD and the siroheme syn- the constrictions are strictly hydrophobic and largely con-
thase CysG (Peakman et  al., 1990). Cole and coworkers served within the FNT family, forming a barrier that must
investigated the transport mechanism of E. coli NirC in be very difficult to overcome for an ionic species. A single
vivo and presented data to support passive nitrite trans- nonhydrophobic side chain is involved in this particular
location as a channel, as well as an active transporter
mode that was suggested to be NO2−/H+ symport (Jia et al.,
2009). The first functional analysis based on isolated
protein reconstituted into proteoliposomes was carried
out by electrophysiology on SSMs, indicating that S. typh-
imurium NirC is able to transport both nitrate and nitrite
anions passively (Rycovska et  al., 2012). From a fluores-
cence assay based on acridine orange that was originally
devised to identify ion extrusion systems (Rosen, 1986),
the authors concluded that NirC additionally acts as a
NO2−/H+ antiporter. In a physiological context, this hypoth-
esis is not straightforward to reconcile with a physiologi-
cal function as a nitrite importer for cytoplasmic reduction
by NirBD: should an antiport mechanism be in place, the
inward-directed proton gradient across the cytoplasmic
membrane would indeed result in active expulsion of
nitrite from the cell. Figure 4 The conducting pore of S. typhimurium NirC.
The stereo representation shows the periplasmic side (p) on top,
NirC is the latest FNT channel for which structural
the cytoplasmic side (c) at the bottom, and the channel as a dotted
information has become available with the most recent surface calculated with HOLE (Smart et al., 1996). Two constric-
crystallographic analysis of the S. typhimurium protein tions created by conserved hydrophobic residues separate a central
at a resolution of 2.4 Å (Lü et  al., 2012b). It follows the vestibule where H197 is located as the single protonable amino acid.

Figure 5 Proposed modes of action for FNT family channels.

(A) The architecture of the conduction pore with a central, hydrophobic vestibule, separated from both the periplasmic and the cytoplas-
mic side by narrow constrictions, is highly conserved. (B) A three-step proton relay involving a conserved histidine in the central vestibule
region, a threonine hydroxyl group, and a coordinated water molecule allows for transient protonation of a monovalent anion that can
overcome the central vestibule as an uncharged species. In this mode, FNT proteins act as bidirectional channels. (C) At low external pH –
as during mixed-acid fermentation in case of FocA – reprotonation of the conserved histidine may occur before the transported acid is
deprotonated. Dissociation will then happen in the cytoplasm, leading to a net translocation of the anion with a proton. This constitutes
secondary active import driven by the proton motive force.

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720      W. Lü et al.: Formate/nitrite transporter family of anion channels
arrangement, and this invariant histidine residue (H197 in
NirC) is essential for transport.
Based on the structure of S. typhimurium NirC, a
mechanism was suggested that reconciles these struc-
tural features with available biochemical and electro-
physiological data (Lü et  al., 2012b). Herein, the hydro-
phobic barrier formed by the two constrictions sealing off
the central vestibule can only be overcome by uncharged
species, and these are obtained by the protonation of
monovalent anions. Consequently, a suitable, (de)pro-
tonable residue is required at each constriction. On the
periplasmic side, this role is played by H197. Its imidazole
side chain can form a stable imidazolium cation that in
the apolar environment of the protein matrix can have a
sufficiently low pKa to transfer a proton to a nitrite anion
at the periplasmic constriction. The uncharged nitrous
acid molecule can then transverse the central vestibule
and pass the cytoplasmic constriction. Here, a coordi-
nated water molecule was observed in the NirC structure,
providing the sole protonable site in the immediate vicin-
ity. From these structural features, a mechanistic model
was derived that involves the cyclic movement of a single
proton around the two restriction sites (Figure 5B). This
implies that the channel is in a transport-competent state
only if H197 is present in its protonated imidazolium
form, and the single available proton in this arrange-
ment is the reason why FNT-mediated ion translocation
is strictly limited to monovalent anions. If during trans-
port the proton is lost to the surrounding medium on
either side, reprotonation will be required, and due to the
existing concentration gradient of H+ across the bacterial
cytoplasmic membrane, this is likely to occur from the
extracellular side (Figure 5C). In consequence, the proton
cycling mechanism of FNT ion translocation accommo-
dates the possibility for a functional switch to second-
ary active proton/anion symport simply by changing
from proton recycling to a vectorial translocation along
the electrochemical proton gradient (Lü et  al., 2012b).
Such a mode of transport would be electroneutral and
thus not amenable to electrophysiological studies that
measure net transport of charge, so that direct biochemi-
cal evidence for the existence of active ion translocation
remains to be provided.
For passive anion permeation, however, electrophysi-
ology has been the method of choice to study FNT channels,
with the first investigations carried out on S. typhimurium
FocA (Lü et al., 2011). Using isolated NirC embedded into
planar lipid bilayer membranes, the specific transport of
nitrite anions through the protein was shown by measur-
ing both macroscopic currents through a series of chan-
nels embedded into a membrane patch and by monitoring
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single-channel events that in addition exhibited distinct
gating behavior (Lü et al., 2012a,b). Currents through NirC
followed Ohm’s law, indicating that gating is not voltage-
dependent, and each single monomer yielded traces that
corresponded to independent opening and closing of the
individual protomer channels. The experimental setup
did not allow assessing the directionality of transport,
as the integration of solubilized NirC pentamers into
liposomes could potentially occur in one of two orienta-
tions, but the data unequivocally established NirC as a
passive channel for nitrite anions (Lü et  al., 2012). This
is in line with the findings from SSM electrophysiology
(Rycovska et  al., 2012), but it did not address the issue
of pH dependence, which in these studies led to the pos-
tulate of nitrite/proton antiport. Furthermore, the SSM
studies also provided the first evidence that nitrate, NO−3,
is transported by NirC as an additional substrate. This
is highly relevant in a physiological context, as nitrate
and nitrite metabolism in enterobacteria such as E. coli
are dependent on a set of enzymes and transporters that
include the nitrate/nitrite antiporters NarK and NarU as
well as NirC as integral membrane proteins and various
redox enzymes on both sides of the cytoplasmic mem-
brane (Jia et al., 2009). Nitrate reductases can be localized
in the periplasm (NapAB) or in the cytoplasmic membrane
facing the cytoplasm (NarGHI, NarZYW). Nitrite reduc-
tases of enterobacteria are either the periplasmic penta-
heme c-type cytochrome NrfA or the assimilatory variant,
the siroheme-dependent NirBD enzyme located in the
cytoplasm (Berks et  al., 1995; Cole, 1996). NirC primar-
ily functions in conjunction with NirBD that is encoded
in the same operon, fulfilling the abovementioned roles
in nitrogen assimilation and detoxification. While nitrite
exported to the periplasm via NirC could be detoxified by
NrfA, there is no obvious reason why nitrite export should
be an active process driven by H+ antiport. NirBD will be
present when NirC is produced, so that export through the
bidirectional NirC should only be relevant if nitrite accu-
mulates very quickly in the cell. Then, however, an active
mechanism should hardly be required because NrfA in the
periplasm is highly active and should suffice to maintain
an outward-directed flow of nitrite.
HSC, a hydrosulfide channel
Only recently, a general phylogenetic analysis of avail-
able FNT family gene sequences revealed the presence
of a distinct subfamily of channels that is most closely
related to NirC. This family was termed FNT3 to denote
 W. Lü et al.: Formate/nitrite transporter family of anion channels      721
the third group of FNT proteins besides FocA/FdhC and
NirC, and its members were located in an operon context
with an assimilatory sulfite reductase that catalyzes the
cytoplasmic six-electron reduction of sulfite, SO3−, to yield
the hydrosulfide anion, HS−, as a toxic product (Czyzewski
and Wang, 2012). A crystal structure of the hydrosulfide
channel AsrD (HSC, FNT3) from Clostridium difficile
revealed the typical pentameric architecture with high
overall similarity to S. typhimurium NirC, documented by
a root mean squared deviation of all atom positions of 0.54
Å for the two structures (Table 1). Contrary to NirC, the
physiological role of AsrD is in exporting its toxic cargo
from the cytoplasm, so that the similarity of both proteins
by itself strongly speaks for bidirectional ion transport in
FNT channels. Through competitive uptake assays, it was
shown that AsrD translocates both formate and nitrite
anions as well as hydrosulfide (Czyzewski and Wang,
2012). In contrast, no significant transport of the divalent
sulfite anion, SO32−, was observed. As for all other FNT
family members, AsrD thus behaves as a low-selectivity
channel for monovalent anions, where the key factor for
hydrosulfide preference is simply the metabolic context
of sulfite reduction in which the protein is produced. This
once more reflects the limited anion selectivity of the bac-
terial membrane and suggests that the expression of fnt
family genes also only occurs in situations when the cor-
responding oxidoreductases are active.
Active and passive transport in the
formate channel FocA
The formate anion, HCOO−, is very similar to nitrite in
structure, size, and pKa, it but plays a very different role
in enterobacterial metabolism. Bulk amounts of formate
are generated by pyruvate:formate lyase, PflB, during the
oxygen-independent mixed-acid fermentation of glucose,
which is abundant in the environment of the gastrointes-
tinal tract (Suppmann and Sawers, 1994). The reaction of
PflB is analogous to one of the pyruvate dehydrogenase
Table 1 Matrix of root mean squared deviations for all atom posi-
tions in known structures of FNT family proteins.
EcFocA (Å) VcFocA (Å) StFocA (Å) StNirC (Å) HSC (Å)
EcFocA 0.51 0.21 2.32 1.20
VcFocA 0.53 1.73 1.06
StFocA 2.40 1.11
StNirC 0.54
HSC
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complex in eukaryotic mitochondria but lacks an oxida-
tive step, so that formate, rather than CO2, is the second
reaction product besides acetyl CoA (Knappe and Sawers,
1990). In total, glucose is converted to two molecules of
formate and two acetyl CoA, which, after further oxida-
tion, are released as acetate, lactate, or ethanol, with
minor amounts of succinate as a by-product. Formate is
exported via the FNT channel FocA, and is subsequently
oxidized to CO2 by a periplasmic formate dehydrogenase
(Leonhartsberger et  al., 2002). The two electrons from
this oxidation reaction are transferred to menaquinone in
the cytoplasmic membrane, and the Q-loop mechanism
that results from the onward electron transfer step leads
to a translocation of two protons from the cytoplasm to
the periplasm. One further proton is released through the
oxidation of HCOO− itself, and consequently, each formate
anion released yields three H+ in the periplasmic space.
Mainly through this mechanism, mixed-acid fermentation
leads to an acidification of the environment that – in batch
culture – is so pronounced that the common metabolic
test for this pathway, the methyl red (MR) test, is merely
an inspection of the pH of the growth medium. The diazo
dye MR undergoes a color transition from yellow to red
with a pKa of 5.1, and the full red color reaction that marks
a growing culture as mixed-acid fermenting is attained at
a pH value below 4.4. This metabolism thus represents a
rapid degradation pathway for glucose in the absence of
oxygen, but it can result in a degree of acidification that
poses a hazard for the enterobacteria themselves. Under
physiological growth conditions, mixed-acid fermenters
thus use a secondary pathway that circumvents the major
proton-releasing step of formate dehydrogenase. When
the pH of the growth medium dropped below a critical
level, Sawers and coworkers found that the export of
formate via FocA ceased (Suppmann and Sawers, 1994).
Instead, the organisms switched to a mode of active –
likely proton-driven – uptake of formate (as formic acid)
and proceeded to convert the compound in a cytoplasmic
disproportionation reaction to CO2 and H2, two gaseous
products that would not accumulate in the medium. The
responsible enzyme complex, formate:hydrogen lyase
(FHL) combines the activities of a formate dehydrogenase
and a hydrogenase and is located on the cytoplasmic side
of the inner membrane (Sawers, 1998). Strikingly, both the
export of formate anions and the import of formic acid
were found to be dependent on the FocA protein, indicat-
ing that the mode of transport switches in a pH-controlled
manner from passive export to secondary active import
(Suppmann and Sawers, 1994). This dual function of a
single protein as channel or transporter in reaction to the
metabolic state is unprecedented and raised considerable
722      W. Lü et al.: Formate/nitrite transporter family of anion channels

interest in the characterization of the membrane protein the N-termini of four protomers were well ordered (Lü
in molecular detail (Sawers, 2005). et  al., 2011). They attained two different conformations,
Of the three structures of FocA known to date, the termed closed and intermediate, with the direct interac-
orthologues from E. coli (Wang et al., 2009) and V. cholerae tion of two neighboring protomer termini, whereas in the
(Waight et al., 2010) were crystallized at high pH, represent- fifth monomer, this part of the structure remained disor-
ing the state where the protein acts as a passive channel dered (Figure 6A–C). Due to fortuitous crystal packing
for formate export from the cytoplasm. In contrast, S. typh- of two FocA pentamers, the asymmetric arrangement of
imurium FocA was crystallized at pH 4.0, so that this state termini was well defined in the two copies of the trans-
should represent the active uptake conformation (Lü et al., porter found in the asymmetric unit, and in both cases, the
2011). The most obvious differences between these struc- sequence of conformers within each pentamer was identi-
tures are in the N-termini of the protomers. E. coli FocA cal (Figure 6D). Besides the N-terminal helix, the only part
yielded diffracting crystals only after the truncation of 22 of FocA that rearranged in the three conformers was the
N-terminal residues that were predicted to form a helical loop region connecting helices TM2b and TM3 that was
stretch (Wang et al., 2009). In V. cholerae FocA, where no previously designated the Ω-loop due to its shape remi-
such truncation was made, this part was disordered and niscent of the Greek letter (Waight et  al., 2010). The pH-
therefore not defined in the electron density map as well dependent conformational switch observed in the crystal
(Waight et al., 2010). In S. typhimurium at low pH, however, structure of S. typhimurium FocA thus coincides very well

A D

Open

Intermediate
E F
C 80 cis: 200 mM HCOO-
trans: 200 mM HCOO-
1.0
60
Relative permeability

40 0.8
20 0.6
/ (pA)

0
0.4
-20
-40 0.2
-60
0
-100 -50 0 50 100 5.1 5.6 6.0 6.5 7.0 7.4
Closed V (mV) pH

Figure 6 Conformational gating in the low-pH form of S. typhimurium FocA.

When crystallized at pH 4.0, the protomers of the FocA pentamer showed three different conformations, differing predominantly in the posi-
tioning of an N-terminal helix, and a connecting loop. The protomers were found in an open (A), intermediate (B), or closed (C) state, and
the arrangement was strictly ordered within the pentamer (D). The observed sequence was O-C-I-C-I, counting clockwise. (E) Macroscopic
currents in voltage-clamp electrophysiology on FocA reconstituted in planar lipid bilayers showed a linear current-voltage response at pH
7.5 (white) that shifted to the expected reversal potential of -40 mV when a gradient of 20 mm/200 mm formate was applied (black). Upon a
shift of pH to 5.1, the current practically ceased (green). (F) A systematic assessment of pH dependence showed a marked gating event at pH
5.7, indicating a cooperative mechanism.

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 W. Lü et al.: Formate/nitrite transporter family of anion channels      723
with the predicted functional switch from passive export
of formate to active import of formic acid (Sawers, 2005).
Electrophysiological studies showed that passive formate
transport ceases at low pH (Lü et al., 2011) (Figure 6E), and
a more detailed analysis revealed that the currents dis-
appear abruptly at pH 5.7 (Figure 6F), hinting at possible
intersubunit cooperativity in the closing event (Lü et  al.,
2012a). A subsequent molecular dynamics (MD) study on
FocA from S. typhimurium and E. coli then assumed His
209, the conserved histidine in the substrate channel’s
central vestibule, to be the relevant pH sensor (Feng et al.,
2012). Twenty nanoseconds of MD calculations with His
209 in a protonated (low pH) or deprotonated (high pH)
state, respectively, then led to variations in the resulting
conformations of the substrate channel, in that at high
pH, the exterior constriction became wider, whereas at
low pH, the interior constriction opened up. Also, at high
pH, one of the protomers observed to be in the closed state
in S. typhimurium FocA at pH 4 transitioned to the open
state, whereas this did not occur at low pH (Feng et  al.,
2012). These results are very well in line with the experi-
mental results of the crystallographic studies, but they fail
to explain why the available structures of FocA proteins at
neutral and low pH show identical conformations of all
channel constrictions.
The structure of S. typhimurium FocA only showed two
out of five protomers in a closed state (Lü et al., 2011), and
the absence of a measurable current indeed points toward
the transport of the uncharged acid that would formally cor-
respond to H+/HCOO− symport, a secondary active process
driven by the proton motive force. Any other stoichiometry
of anions and protons would result in a net current and
can therefore be excluded at this point. However, no direct
evidence for the transport of formic acid has been provided
for the isolated protein, and understanding the details of
the mechanism will require further studies.
The substrate range of formate
channels
Electrophysiological studies of reconstituted FocA from
S. typhimurium into planar lipid bilayers generated from
E. coli polar lipid extracts yielded an assessment of the
pH-dependent gating mechanism described above (Lü
et  al., 2011, 2012a). Due to the high conductivity of the
channel, it was also possible to measure single-channel
currents and use these to determine the conductance
for formate anions with respect to substrate concentra-
tion. At high pH, FocA showed a maximum conductance
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of Gformate = 26.9 ± 0.5 pS, with an affinity (at half-maximal
conductance) of kformate = 11.7 ± 0.1 mm (Lü et  al., 2012a).
When FocA was initially described, it was found to
conduct not only formate, but also the structurally
similar hypophosphite anion, H2PO2− (Suppmann and
Sawers, 1994). In the course of an electrophysiological
characterization of S.  typhimurium FocA, chloride ions
showed the same permeability through FocA as formate
and hypophosphite (Lü et  al., 2012a). Anion channels
are frequently less selective for their transported species
than cation channels, and consequently, a broad search
for other candidates was initiated. Consistently, FocA did
not transport divalent or polyvalent anions, but at the
same time, its selectivity among monovalent anions was
surprisingly low (Figure 7). Besides formate, hypophos-
phite, and chloride, FocA efficiently transported nitrite
and also lactate, acetate, and pyruvate with relative per-
meabilities under bi-ionic conditions that were at least
40% of the one for formate (Table  2) (Lü et  al., 2012a).
Single-channel currents for these alternative sub-
strates were recorded as well, yielding an unexpected
Figure 7 FNT channels transport a wide range of monovalent
anions.
Different known cargo molecules underline that specific recognition
and translocation that strictly exclude leakage of water or
protons requires a substantial degree of flexibility within the
conduction pore. FocA was originally characterized as a channel
for hypophosphite and formate, whereas AsrD was a hydrosulfide
channel. Nitrate transport was observed on SSM vesicles
with S. typhimurium NirC. All other molecules were identified
as transported species through direct electrophysiology on
S. typhimurium FocA and NirC.
Table 2 Relative permeabilities and conductance parameters for
StFocA for various anions.
Relative Maximum Affinity k (mm)
permeability conductance G (pS)
Formate 1.00 26.9 ± 0.5 11.7 ± 0.1
Acetate 0.43 ± 0.08 26.2 ± 0.5 23.9 ± 1.7
Lactate 0.89 ± 0.05 26.5 ± 1.8 96.0 ± 14.4
Pyruvate 0.73 ± 0.05 11.1 ± 0.4 11.6 ± 1.9
Chloride 1.03 ± 0.1 6.8 ± 0.5 21.9 ± 5.1
724      W. Lü et al.: Formate/nitrite transporter family of anion channels
distribution of maximum conductances G and affinities k
(Table 2). Although acetate, lactate, and pyruvate showed
40%–90% of the permeability of formate, only acetate
and lactate were actually conducted well, with Gmax
values that were nearly identical to the one for formate.
Pyruvate was conducted with a lower Gmax, and the same
was true for chloride that under bi-ionic conditions had
exhibited the same relative permeability as formate.
Thus, although both pyruvate and chloride bound well to
FocA, they were not efficiently translocated. In contrast,
formate, acetate, and lactate were efficiently transported,
and most notably, these three molecules are the main
anionic products of the metabolic pathway of mixed-acid
fermentation. FocA is expressed from an operon with
PflB, the enzyme that splits pyruvate into formate and
acetyl CoA. The fate of acetyl CoA is then to be converted
to acetate via acetyl phosphate or to ethanol. Both mole-
cules are exported from the cell. Additional relevant met-
abolic flux is through homolactic fermentation of pyru-
vate to yield lactate that is exported from the cell as well.
The range of monovalent anions transported by FocA
matches the product spectrum of mixed-acid fermenta-
tion very well, and given the abundance of the anions
formate, acetate, and lactate under these conditions and
the fact that no alternative, dedicated carriers for the
latter two are known, render FocA a universal exporter
for the products of mixed-acid fermentation under physi-
ological conditions (Lü et al., 2012a). Moreover, although
the three anions are transported with near identical con-
ductances Gmax (Table 2), their respective affinities cor-
respond to the relative abundance of products derived
from metabolic fluxes during mixed-acid fermentation
in E. coli as determined in chemostat experiments (Yang
et al., 2001). The authors found that only 3.4% of pyru-
vate were reduced to lactate in homolactic fermentation,
whereas the bulk would be a substrate for PflB, generat-
ing formate and acetyl CoA. A total of 49.6% of the latter
was then converted to acetate, the remainder to ethanol.
Under these conditions, mixed-acid fermentation thus
yielded formate, acetate, and lactate at an approximate
ratio of 1:0.5:0.04, whereas the affinities of FocA were
determined to a ratio of 1:0.5:0.11 (Table 2). FocA thus
presents itself as an optimized channel for the anionic
products of mixed-acid fermentation. Its additional,
proposed functionality as a secondary active transport
protein further adds to its role as a key player in entero-
bacterial energy metabolism. The mechanism outlined
for NirC above provides a rationale for the bidirectional
passive translocation of monovalent anions (Figure 5B)
and also for a switch to secondary active import at low
external pH (Figure 5C). Although this situation may not
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be of relevance for NirC, it quite precisely matches the
physiological data for FocA.
Functional adaptations differentiate
FNT channels
Judging from the available high-resolution structures
of NirC, AsrD, and FocA, the structural properties of the
protomer substrate channel and the selectivity pore with
two hydrophobic constrictions separating a central ves-
tibule lined by an invariant histidine residue are highly
conserved among all FNT family channels. The topologi-
cal profiles of the transport channels are very similar,
and all FNT channels conduct at least nitrite and formate
anions. Nevertheless, all three channel types have largely
divergent metabolic roles (Figure 1), with the additional
feature of a functional switch to active transport as pos-
tulated for FocA. Also, the conductances and affinities
for different anions vary between FocA and NirC, with the
first preferring formate over nitrite and vice versa for NirC
(Lü et  al., 2012a,b). The basis for this difference may be
found in the electrostatic properties of the FNT pentamer
surfaces that face the periplasm and cytoplasm, respec-
tively. Most integral membrane proteins of the bacterial
cytoplasmic membrane follow the ‘positive-inside’ rule
(von Heijne and Gavel, 1988), stating that during Sec-
dependent membrane insertion of the proteins, positively
charged regions are retained in the cytoplasm, leading
to an overall positive charge of the cytoplasmic face of
the folded membrane protein (Figure 8). FocA, NirC, and
AsrD adhere to this scheme, with NirC showing the inter-
esting variation of having a negatively charged patch at
the cytoplasmic entrance of the ion channel (Lü et  al.,
2012b). In contrast, there is less homogeneity in the
electrostatic surface charge distribution on the periplas-
mic face. Both NirC and AsrD show positive electrostatic
surface potential distributions, in particular, around the
entrance funnels to the ion channels (Figure 8B and C).
The two proteins are thus well constructed to function
bidirectionally for anions that will be attracted toward
the conducting pores following the electrostatic field gra-
dient. Although the directionality of transport has so far
not been studied in either system, it can be assumed that
the physiological roles of nitrite import vs. hydrosulfide
export are mainly based on the availability of the anions.
Nitrite is encountered as a substrate in the periplasm and
is quickly reduced by NirBD nitrite reductase in the cyto-
plasm, so that the concentration gradient will always be
directed inward. In contrast, hydrosulfide is the product
 W. Lü et al.: Formate/nitrite transporter family of anion channels      725
Figure 8 Surface charge distribution as a determinant of FNT
channel selectivity.
Although the transport channel is highly conserved, the physi-
ological function of different channels is affected by the electro-
static surface potential on the cytoplasmic and periplasmic faces.
(A) Cytoplasmic and periplasmic faces of S. typhimurium FocA.
The channel acts as an anion exporter and the negative surface
potential on the periplasmic side counteracts anion influx. (B) The
hydrosulfide channel HSC/AsrD is an anion exporter and operates
under conditions of an outward directed hydrosulfide gradient.
(C) S. typhimurium NirC, in contrast, is an anion importer, and the
action of nitrite reductase NirBD that is encoded in the same operon
leads to an inward directed nitrite gradient. Both AsrD and NirC thus
function as bidirectional channels, and the differences in transport
direction are created by the existing anion gradients.
of cytoplasmic sulfite reduction and will be dissipated to
the environment. Its concentration gradient will conse-
quently be directed outward.
A different situation is encountered in FocA, where the
protein shows a distinctly negative surface potential dis-
tribution on the periplasmic face, energetically disfavor-
ing the import of a negatively charged species (Figure 8A).
Again, this is well in line with the role of FocA in mixed-
acid fermentation: although the symmetric selectivity
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filter in the channels is fully bidirectional, the combina-
tion of a positively charged inside face and the negatively
charged periplasmic side results in a preference for anion
export. This exactly meets the requirements of mixed-acid
fermentation at high external pH when formate is to be
exported, and it even allows for a limited accumulation of
the metabolite in the periplasm without significant back-
flow into the cell. This should be beneficial, as the redox
enzyme FDH depends on the availability of menaquinone
as an electron acceptor, and this process is likely slower
than formate generation from glucose through glycolysis
and PflB. Conversely, when extracellular pH starts to drop
and FocA switches to an active uptake mode, the nega-
tive surface charge is no longer a hindrance. Not only may
some surface residues become protonated under such
conditions, but also the imported species itself is not
the formate anion, but uncharged formic acid, HCOOH.
Protein electrostatics thus play no role, and the proton
that drives the symport mechanism may well be the one on
formic acid. It would then be the conformational changes
of the N-terminal helices that hinder the efflux of formic
acid, while, in addition, the intracellular concentration of
the species is kept very low, as it is continuously dispro-
portionated by the FHL complex.
In conclusion, monovalent anion channels of the FNT
family are versatile and efficient translocators with mod-
erately low specificity for the transported species. They
share a common ancestry with aquaporins and glycerop-
orins but have developed an efficient selectivity filter that
is based on combining two distinct transport routes for an
anion and a single proton. The size range of transported
ions points toward considerable flexibility within the
transport channel, and future studies should be directed
toward depicting the different structural states associated
with the transport process.
Acknowledgements: The authors thank the staff at beam
lines X06DA and X06SA at the Swiss Light Source, Vil-
ligen, Switzerland, for their continuous support during
data collection and Elke Gerbig-Smentek for the excel-
lent technical assistance. This work was supported by
Deutsche Forschungsgemeinschaft [grants An 676/1 and
An 676/3 to S.L.A.A., grants Ei 520/3 and Ei 520/6 (RU 929)
to O.E., and IRTG 1478].
Received November 26, 2012; accepted February 4, 2013; previously
published online February 5, 2013
726      W. Lü et al.: Formate/nitrite transporter family of anion channels
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