Charge Translocation Research Articles

Experimentally optimized threading structures of the proton-coupled folate transporter.

The proton‐coupled folate transporter (PCFT, SLC46A1) transports folic acid across the plasma membrane, together with an excess of protons such that the net charge translocation is positive. We developed 3D structural models of PCFT threaded onto the X‐ray structures of major facilitator superfamily (MFS) members that were identified as close structural homologues. The model of PCFT threaded onto the glycerol‐3‐phosphate transporter (GlpT) structure is consistent with detailed accessibility studies in the absence of extracellular substrate and at pH 7.4 presented here, and additionally with a multitude of other mutagenesis and functional studies. Characteristic MFS structural features are preserved in this PCFT model, such as 12 transmembrane helices divided into two pseudosymmetric bundles, and a high density of positive charges on the periphery of the cytoplasmic site that allow interactions with negatively charged lipid head‐groups. Under the experimental conditions, PCFT predominantly samples the resting state, which in this case is inward‐open. Several positions lining the substrate cavity have been identified. Motif A, a helix‐turn‐helix motif that is a hallmark of MFS transporters between transmembrane segments II and III is oriented appropriately to interact with residues from transmembrane segments IV as well as XI upon conformational transition to the outward‐open state. A charge‐relay system between three charged residues as well as apposing glycines in two α‐helices, both contributed to by motif A, become engaged when PCFT is modeled on the outward‐open state of a putative proton‐driven transporter (YajR).

Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology.

Organophosphorus compounds (OPC), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). Inhibited AChE results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nAChR) in the postsynaptic membrane is provoked. Direct targeting of nAChR to reduce receptor desensitisation might be an alternative therapeutic approach. For drug discovery, functional properties of potent therapeutic candidates need to be investigated in addition to affinity properties. Solid supported membrane (SSM)-based electrophysiology is useful for functional characterisation of ligand-gated ion channels like nAChRs, as charge translocations via capacitive coupling of the supporting membrane can be measured. By varying the agonist (carbamoylcholine) concentration, different functional states of the nAChR were initiated. Using plasma membrane preparations obtained from Torpedo californica electric organ, functional properties of selected nAChR ligands and non-oxime bispyridinium compounds were investigated. Depending on overall-size, the bispyridinium compounds enhanced or inhibited cholinergic signals induced by 100 μM carbamoylcholine. Applying excessive concentrations of the agonist carbamoylcholine provoked desensitisation of the nAChRs, whereas addition of bispyridinium compounds bearing short alkyl linkers exhibited functional recovery of previously desensitised nAChRs. The results suggest that these non-oxime bispyridinium compounds possibly interacted with nAChR subtypes in a manner of a positive allosteric modulator (PAM). The described newly developed functional assay is a valuable tool for the assessment of functional properties of potential compounds such as nAChR modulating ligands, which might be a promising approach in the therapeutically treatment of OPC-poisonings.

Computational Characterization of Conformational Transitions in the Voltage-Sensing Domain of Ci-VSP

The canonical voltage-sensing domain (VSD) is a conserved structural module that transduces the electric field across cellular membranes into defined protein motions of its charge-rich helix, the S4 segment. Up on depolarization, the VSD rearranges from the ‘resting’ (or ‘down’) state conformation into the ‘active’ (or ‘up’) state conformation, which in turn regulates the motion of downstream modules of the protein, such as opening of the pore domain of a voltage-gated ion channel or the activity of an enzyme. The voltage-sensing process has long attracted much interest and fruitful results have been reported using a variety of functional, spectroscopic and structural approaches. Recently, we solved crystal structures of the VSD of the Ciona intestinalis voltage-sensing phosphatase Ci-VSP in both it resting (Down) and activated (Up) conformations. Comparison of the two structures reveals an ∼ 5-A three-residue displacement of the S4 segment along, together with an ∼ 60° rotation around, its axis (called the one-click gating motion), while the S1-S3 segments remain fairly stable. Using 2-D self-learning umbrella sampling molecular dynamics simulations, combined with homology modeling and the string method, we have now calculated the free energy landscape governing the conformational changes of Ci-VSD. These calculations have allowed us to explore the energetic viability of further up (one-click up) and further down (two-clicks down) conformations, as potential mechanisms for additional charge translocation. These conformations should advance our understanding of the detailed mechanisms of voltage-gating and allow for the design of new experiments aimed to verify the present model of S4 movements and gating charge translocation.

Computationally Efficient Multiscale Reactive Molecular Dynamics to Describe Amino Acid Deprotonation in Proteins.

An important challenge in the simulation of biomolecular systems is a quantitative description of the protonation and deprotonation process of amino acid residues. Despite the seeming simplicity of adding or removing a positively charged hydrogen nucleus, simulating the actual protonation/deprotonation process is inherently difficult. It requires both the explicit treatment of the excess proton, including its charge defect delocalization and Grotthuss shuttling through inhomogeneous moieties (water and amino residues), and extensive sampling of coupled condensed phase motions. In a recent paper (J. Chem. Theory Comput.2014, 10, 2729−273725061442), a multiscale approach was developed to map high-level quantum mechanics/molecular mechanics (QM/MM) data into a multiscale reactive molecular dynamics (MS-RMD) model in order to describe amino acid deprotonation in bulk water. In this article, we extend the fitting approach (called FitRMD) to create MS-RMD models for ionizable amino acids within proteins. The resulting models are shown to faithfully reproduce the free energy profiles of the reference QM/MM Hamiltonian for PT inside an example protein, the ClC-ec1 H+/Cl– antiporter. Moreover, we show that the resulting MS-RMD models are computationally efficient enough to then characterize more complex 2-dimensional free energy surfaces due to slow degrees of freedom such as water hydration of internal protein cavities that can be inherently coupled to the excess proton charge translocation. The FitRMD method is thus shown to be an effective way to map ab initio level accuracy into a much more computationally efficient reactive MD method in order to explicitly simulate and quantitatively describe amino acid protonation/deprotonation in proteins.

Stepwise vs concerted excited state tautomerization of 2-hydroxypyridine: Ammonia dimer wire mediated hydrogen/proton transfer.

The stepwise and concerted excited state intermolecular proton transfer (PT) and hydrogen transfer (HT) reactions in 2-hydroxypyridine-(NH3)2 complex in the gas phase under Cs symmetry constraint and without any symmetry constraints were performed using quantum chemical calculations. It shows that upon excitation, the hydrogen bonded in 2HP-(NH3)2 cluster facilitates the releasing of both hydrogen and proton transfer reactions along ammonia wire leading to the formation of the 2-pyridone tautomer. For the stepwise mechanism, it has been found that the proton and the hydrogen may transfer consecutively. These processes are distinguished from each other through charge translocation analysis and the coupling between the motion of the proton and the electron density distribution along ammonia wire. For the complex under Cs symmetry, the excited state HT occurs on the A″((1)πσ*) and A'((1)nσ*) states over two accessible energy barriers along reaction coordinates, and excited state PT proceeds mainly through the A'((1)ππ*) and A″((1)nπ*) potential energy surfaces. For the unconstrained complex, potential energy profiles show two (1)ππ*-(1)πσ* conical intersections along enol → keto reaction path indicating that proton and H atom are localized, respectively, on the first and second ammonia of the wire. Moreover, the concerted excited state PT is competitive to take place with the stepwise process, because it proceeds over low barriers of 0.14 eV and 0.11 eV with respect to the Franck-Condon excitation of enol tautomer, respectively, under Cs symmetry and without any symmetry constraints. These barriers can be probably overcome through tunneling effect.

Respiratory conservation of energy with dioxygen: cytochrome C oxidase.

Cytochrome c oxidase (CcO) is the terminal oxidase of cell respiration which reduces molecular oxygen (O₂) to H2O coupled with the proton pump. For elucidation of the mechanism of CcO, the three-dimensional location and chemical reactivity of each atom composing the functional sites have been extensively studied by various techniques, such as crystallography, vibrational and time-resolved electronic spectroscopy, since the X-ray structures (2.8 Å resolution) of bovine and bacterial CcO have been published in 1995.X-ray structures of bovine CcO in different oxidation and ligand binding states showed that the O₂reduction site, which is composed of Fe (heme a 3) and Cu (CuB), drives a non-sequential four-electron transfer for reduction of O₂to water without releasing any reactive oxygen species. These data provide the crucial structural basis to solve a long-standing problem, the mechanism of the O₂reduction.Time-resolved resonance Raman and charge translocation analyses revealed the mechanism for coupling between O₂reduction and the proton pump: O₂is received by the O₂reduction site where both metals are in the reduced state (R-intermediate), giving the O₂-bound form (A-intermediate). This is spontaneously converted to the P-intermediate, with the bound O₂fully reduced to 2 O²⁻. Hereafter the P-intermediate receives four electron equivalents from the second Fe site (heme a), one at a time, to form the three intermediates, F, O, and E to regenerate the R-intermediate. Each electron transfer step from heme a to the O₂reduction site is coupled with the proton pump.X-ray structural and mutational analyses of bovine CcO show three possible proton transfer pathways which can transfer pump protons (H) and chemical (water-forming) protons (K and D). The structure of the H-pathway of bovine CcO indicates that the driving force of the proton pump is the electrostatic repulsion between the protons on the H-pathway and positive charges of heme a, created upon oxidation to donate electrons to the O₂reduction site. On the other hand, mutational and time-resolved electrometric findings for the bacterial CcO strongly suggest that the D-pathway transfers both pump and chemical protons. However, the structure for the proton-gating system in the D-pathway has not been experimentally identified. The structural and functional diversities in CcO from various species suggest a basic proton pumping mechanism in which heme a pumps protons while heme a 3 reduces O₂as proposed in 1978.

Functional analysis of human Na(+)/K(+)-ATPase familial or sporadic hemiplegic migraine mutations expressed in Xenopus oocytes.

Functional characterization of ATP1A2 mutations that are related to familial or sporadic hemiplegic migraine (FHM2, SHM). cRNA of human Na(+)/K(+)-ATPase α2- and β1-subunits were injected in Xenopus laevis oocytes. FHM2 or SHM mutations of residues located in putative α/β interaction sites or in the α2-subunit's C-terminal region were investigated. Mutants were analyzed by the two-electrode voltage-clamp (TEVC) technique on Xenopus oocytes. Stationary K(+)-induced Na(+)/K(+) pump currents were measured, and the voltage dependence of apparent K(+) affinity was investigated. Transient currents were recorded as ouabain-sensitive currents in Na(+) buffers to analyze kinetics and voltage-dependent pre-steady state charge translocations. The expression of constructs was verified by preparation of plasma membrane and total membrane fractions of cRNA-injected oocytes. Compared to the wild-type enzyme, the mutants G900R and E902K showed no significant differences in the voltage dependence of K(+)-induced currents, and analysis of the transient currents indicated that the extracellular Na(+) affinity was not affected. Mutant G855R showed no pump activity detectable by TEVC. Also for L994del and Y1009X, pump currents could not be recorded. Analysis of the plasma and total membrane fractions showed that the expressed proteins were not or only minimally targeted to the plasma membrane. Whereas the mutation K1003E had no impact on K(+) interaction, D999H affected the voltage dependence of K(+)-induced currents. Furthermore, kinetics of the transient currents was altered compared to the wild-type enzyme, and the apparent affinity for extracellular Na(+) was reduced. The investigated FHM2/SHM mutations influence protein function differently depending on the structural impact of the mutated residue.

Struktur und Funktion der mitochondrialen Atmungskettenkomplexe

The molecular mechanisms of redox driven proton pumps are surprisingly diverse. Studying them is a great challenge, since protons are also involved in acid/base equilibria and many other redox reactions. Indirect pumping by long range interactions in complex I, ligand conduction via bifurcated electron transfer in complex III and direct proton pumping combined with charge translocation by an incomplete redox loop in complex IV are found in the respirasomes of the mitochondrial respiratory chain.

LETM1 in mitochondrial cation transport.

LETM1 in mitochondrial cation transport.

Evolutionary Perspective on the Coupling Mechanism of Complex I and Related Enzymes

Complex I is an energy transducing enzyme present in the three domains of life. This enzyme catalyzes the oxidation of NADH and the reduction of quinone coupled to charge translocation across the membrane. In this way, it contributes to the establishment of the transmembrane difference of electrochemical potential which is used for ATP synthesis, solute transport and motility. The research on this enzyme has gained a new enthusiasm, especially after the resolution of the crystallographic structures of bacterial and mitochondrial complexes. Most attention is now dedicated to the investigation of the energy coupling mechanism. In this work, we made a thorough investigation of complex I and group 4 [NiFe] hydrogenases and established a third member of this family of proteins: the energy-converting hydrogenase related complex. We observed that four subunits (NuoB, D, H and antiporter-like) are common to the 3 types of complexes and we have denominated these subunits as the universal adaptor. We further explored the properties of the adaptor by investigating the structural characteristics of the antiporter-like subunit. We observed that the adaptor contains an antiporter-like subunit with a long amphipathic α-helix. The long helix is a common denominator that has been conserved through evolution. This should reflect a key role of such helix in the coupling mechanism of this family of enzymes. We are currently investigating the structural motifs involved in Na+/H+ antiporter activity in complex I and related complexes. These findings are a step forward in the investigation of the coupling mechanism of complex I.

Sarcoplasmic Reticulum K+ (TRIC) Channel Does Not Carry Essential Countercurrent during Ca2+ Release

The charge translocation associated with sarcoplasmic reticulum (SR) Ca2+ efflux is compensated for by a simultaneous SR K+ influx. This influx is essential because, with no countercurrent, the SR membrane potential (Vm) would quickly (<1 ms) reach the Ca2+ equilibrium potential and SR Ca2+ release would cease. The SR K+ trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K+ countercurrent during release. To better define the physiological role of the SR K+ channel, we compared SR Ca2+ transport in saponin-permeabilized cardiomyocytes before and after limiting SR K+ channel function. Specifically, we reduced SR K+ channel conduction 35 and 88% by replacing cytosolic K+ for Na+ or Cs+ (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca2+ reloading, and caffeine-evoked Ca2+ release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K+ (TRIC) channels is not required to support SR Ca2+ release (or uptake). Because K+ enters the SR through RyRs during release, the SR K+ (TRIC) channel most likely is needed to restore trans-SR K+ balance after RyRs close, assuring SR Vm stays near 0 mV.

Photoinduced Proton Coupled Electron Transfer in 2-(2′-Hydroxyphenyl)-Benzothiazole

We characterize the structural and electronic changes during the photoinduced enol-keto tautomerization of 2-(2'-hydroxyphenyl)-benzothiazole (HBT) in a nonpolar solvent (tetrachloroethene). We quantify the redistribution of electronic charge and intramolecular proton translocation in real time by combining UV-pump/IR-probe spectroscopy and quantum chemical modeling. We find that the photophysics of this prototypical molecule involves proton coupled electron transfer (PCET), from the hydroxyphenyl to the benzothiazole rings, resulting from excited state intramolecular proton transfer (ESIPT) coupled to electron transfer through the conjugated double bond linking the two rings. The combination of polarization-resolved mid-infrared spectroscopy of marker modes and time-dependent density functional theory (TD-DFT) provides key insights into the transient structures of the molecular chromophore during ultrafast isomerization dynamics.

Voltage-dependent processes in the electroneutral amino acid exchanger ASCT2

Neutral amino acid exchange by the alanine serine cysteine transporter (ASCT)2 was reported to be electroneutral and coupled to the cotransport of one Na+ ion. The cotransported sodium ion carries positive charge. Therefore, it is possible that amino acid exchange is voltage dependent. However, little information is available on the electrical properties of the ASCT2 amino acid transport process. Here, we have used a combination of experimental and computational approaches to determine the details of the amino acid exchange mechanism of ASCT2. The [Na+] dependence of ASCT2-associated currents indicates that the Na+/amino acid stoichiometry is at least 2:1, with at least one sodium ion binding to the amino acid–free apo form of the transporter. When the substrate and two Na+ ions are bound, the valence of the transport domain is +0.81. Consistently, voltage steps applied to ASCT2 in the fully loaded configuration elicit transient currents that decay on a millisecond time scale. Alanine concentration jumps at the extracellular side of the membrane are followed by inwardly directed transient currents, indicative of translocation of net positive charge during exchange. Molecular dynamics simulations are consistent with these results and point to a sequential binding process in which one or two modulatory Na+ ions bind with high affinity to the empty transporter, followed by binding of the amino acid substrate and the subsequent binding of a final Na+ ion. Overall, our results are consistent with voltage-dependent amino acid exchange occurring on a millisecond time scale, the kinetics of which we predict with simulations. Despite some differences, transport mechanism and interaction with Na+ appear to be highly conserved between ASCT2 and the other members of the solute carrier 1 family, which transport acidic amino acids.

Cell-Free Electrophysiological Functional Measurements with Native Membranes of Torpedo Californica Electric Tissue

Objective: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels which mediate Na+, K+ and Ca2+ conductance. The human muscle subtype α1β1δe is an important target in drug discovery related to anaesthesia or therapeutic approaches of intoxications with organophosphorus compounds. Because of its high degree of homology with the human muscle subtype, αβδγ nAChRs from the Torpedo electric organ are a suitable surrogate. For functional characterisation, cell-free electrophysiological methods based on solid supported membranes (SSM) can be useful in cases where conventional electrophysiology cannot be applied. In this sensor based technique, charge translocation is measured via capacitive coupling of the supporting membrane. To obtain a sufficient current density, multiple parameters have an impact evaluated in this study.Experimental procedures: Crude membranes from frozen electric organ of Torpedo californica were purified with sucrose gradient centrifugation and then adsorbed to SSM varying buffers, average vesicle size and incubation. The activation of nAChR was measured with a SURFE2R workstation allowing automated high throughput measurements. Voltage dependent sodium channels were blocked with ambroxol. Rapid exchange of a non-activating buffer to an activating buffer containing the agonist carbamoylcholine initiated Na+ influx in the membrane vesicles. The results were correlated with the different experimental designs.Results: Depending on the average particle size, zeta potential and incubation, stable and reproducible cholinergic signals were recorded (∼ 1 nA). The use of ambroxol prevented a premature collapse of the sodium gradient. Increasing carbamoylcholine content in the activating solution led to curves with saturation behaviour. However, carbamoylcholine concentrations > 1 mM seemed to induce desensitisation of the receptors.Conclusion: The results presented here demonstrate that functional high-throughput measurements can be performed with native membranes of Torpedo californica electric tissue. Though, maintaining and regeneration of sodium gradient requires sensitive measurements.

Steps of the coupled charge translocation in the catalytic cycle of cytochrome c oxidase

Cytochromecoxidase (COX) terminates the respiratory chain in mitochondria and in plasmatic membrane of many aerobic bacteria. The enzyme reduces dioxygen molecule into water and the reaction is accompanied with generation of transmembrane difference of electric potentials. The energy conservation by COX is based on the vectorial organization of the chemical reaction due to substrate protons transfer from the negative phase into the active site to meet the electrons, coming from opposite side of the membrane. In addition, a half of free energy of redox-chemistry reaction is conserved through transmembrane proton pumping. Each of the reaction steps in the catalytic cycle of COX involves a sequence of coupled electron and proton transfer reaction. The time-resolved study of the coupled charge translocation during catalytic cycle of cytochromecoxidase is reviewed. Based on many fascinating parallels between the mechanisms of COX and pigment-protein complex of photosystem II from oxygenic photosynthetic organisms, the data described could be used in the light-driven water-splitting process.

Charge Compensation Mechanism of a Na+-coupled, Secondary Active Glutamate Transporter

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.

Proton transfer reactions in carbon nanotubes endohedrally functionalized with selected polar amino acid sidechains

We use the empirical-valence-bond (EVB) theory to investigate intramolecular proton transfer reactions between a selected set of polar amino acid sidechains and hydroxyl groups suspended inside carbon nanotubes to model the effect of hydrophobic confinement on the energetics of proton transfer involving (i) translocation of an excess protonic charge (with protonated histidine sidechain as donor) and (ii) transformation of a neutral reactant state to a charge-separated product state (with sidechains of Asp, Glu, Ser and Thr as donor). In both the cases, confinement in hydrophobic medium is found to change the associated free energies compared to their respective values in the bulk solution phase. Presence of stable hydrogen bonding within the pore is found to have a significant effect on both free energies of reaction and activation and thus governs the thermodynamic and kinetic feasibilities of these intramolecular reactions in hydrophobic confinement.

Excess protons in water-acetone mixtures

Using molecular dynamics experiments, we analyze equilibrium and dynamical characteristics related to the solvation of excess protons in water-acetone mixtures. Our approach is based on the implementation of an extended valence-bond Hamiltonian, which incorporates translocation of the excess charge between neighboring water molecules. Different mixtures have been analyzed, starting from the pure water case down to solutions with a water molar fraction x(w) = 0.25. In all cases, we have verified that the structure of the first solvation shell of the H(3)O(+) moiety remains practically unchanged, compared to the one observed in pure water. This shell is composed by three water molecules acting as hydrogen bond acceptors, with no evidence of hydrogen bond donor-like connectivity. Moreover, the increment in the acetone concentration leads to a gradual stabilization of Eigen-like [H(3)O[middle dot](H(2)O)(3)](+) configurations, in detriment of Zundel-like [H[middle dot](H(2)O)(2)](+) ones. Rates of proton transfer and proton diffusion coefficients have been recorded at various water-acetone relative concentrations. In both cases, we have found a transition region, in the vicinity of x(w) ∼ 0.8, where the concentration dependences of the two magnitudes change at a quantitative level. A crude estimate shows that, at this tagged concentration, the volumes "occupied" by the two solvents become comparable. The origins of this transition separating water-rich from acetone-rich realms is rationalized in terms of modifications operated in the nearby, second solvation shell, which in the latter solutions, normally includes at least, one acetone molecule. Our results would suggest that one possible mechanism controlling the proton transfer in acetone-rich solutions is the exchange of one of these tagged acetone molecules, by nearby water ones. This exchange would give rise to Zundel-like structures, exhibiting a symmetric, first solvation shell composed exclusively by water molecules, and would facilitate the transfer between neighboring water molecules along the resonant complex.

The role of proton and sodium ions in energy transduction by respiratory complex I

Respiratory complex I plays a central role in energy transduction. It catalyzes the oxidation of NADH and the reduction of quinone, coupled to cation translocation across the membrane, thereby establishing an electrochemical potential. For more than half a century, data on complex I has been gathered, including recently determined crystal structures, yet complex I is the least understood complex of the respiratory chain. The mechanisms of quinone reduction, charge translocation and their coupling are still unknown. The H(+) is accepted to be the coupling ion of the system; however, Na(+) has also been suggested to perform such a role. In this article, we address the relation of those two ions with complex I and refer ion pump and Na(+)/H(+) antiporter as possible transport mechanisms of the system. We put forward a hypothesis to explain some apparently contradictory data on the nature of the coupling ion, and we revisit the role of H(+) and Na(+) cycles in the overall bioenergetics of the cell.

The nitrite transport protein NirC from Salmonella typhimurium is a nitrite/proton antiporter

In anaerobically grown bacteria, transport of nitrite is catalyzed by an integral membrane protein of the form ate–nitrite transporter family, NirC, which in Salmonella typhimurium plays a critical role in intracellular virulence. We present a functional characterization of the S. typhimurium nitrite transporter StmNirC in native membrane vesicles as well as purified and reconstituted into proteoliposomes. Using an electrophysiological technique based on solid supported membranes, we show nitrite induced translocation of negative charges into proteoliposomes reconstituted with purified StmNirC. These data demonstrate the electrogenicity of StmNirC and its substrate specificity for nitrite. Monitoring changes in ΔpH on everted membrane vesicles containing overexpressed StmNirC using acridine orange as a pH indicator we demonstrate that StmNirC acts as a secondary active transporter. It promotes low affinity transport of nitrite coupled to H+ antiport with a pH independent profile in the pH range from 6 to 8. In addition to nitrite also nitrate is transported by StmNirC, but with reduced flux and complete absence of proton antiport activity. Taken together, these data suggest a bispecific anion selectivity of StmNirC with an ion specific transport mode. This may play a role in regulating nitrite transport under physiological conditions.