The structural basis of secondary active transport mechanisms

Secondary active transporters couple the free energy of the electrochemical potential of one solute to the transmembrane movement of another. As a basic mechanistic explanation for their transport function the model of alternating access was put forward more than 40 years ago, and has been supported by numerous kinetic, biochemical and biophysical studies. According to this model, the transporter exposes its substrate binding site(s) to one side of the membrane or the other during transport catalysis, requiring a substantial conformational change of the carrier protein. In the light of recent structural data for a number of secondary transport proteins, we analyze the model of alternating access in more detail, and correlate it with specific structural and chemical properties of the transporters, such as their assignment to different functional states in the catalytic cycle of the respective transporter, the definition of substrate binding sites, the type of movement of the central part of the carrier harboring the substrate binding site, as well as the impact of symmetry on fold-specific conformational changes. Besides mediating the transmembrane movement of solutes, the mechanism of secondary carriers inherently involves a mechanistic coupling of substrate flux to the electrochemical potential of co-substrate ions or solutes. Mainly because of limitations in resolution of available transporter structures, this important aspect of secondary transport cannot yet be substantiated by structural data to the same extent as the conformational change aspect. We summarize the concepts of coupling in secondary transport and discuss them in the context of the available evidence for ion binding to specific sites and the impact of the ions on the conformational state of the carrier protein, which together lead to mechanistic models for coupling.     

Unbiased simulations reveal the inward-facing conformation of the human serotonin transporter and Na(+) ion release

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 μs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na(+)-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport.     

Unifying concept of serotonin transporter-associated currents

Serotonin (5-HT) uptake by the human serotonin transporter (hSERT) is driven by ion gradients. The stoichiometry of transported 5-HT and ions is predicted to result in electroneutral charge movement. However, hSERT mediates a current when challenged with 5-HT. This discrepancy can be accounted for by an uncoupled ion flux. Here, we investigated the mechanistic basis of the uncoupled currents and its relation to the conformational cycle of hSERT. Our observations support the conclusion that the conducting state underlying the uncoupled ion flux is in equilibrium with an inward facing state of the transporter with K+ bound. We identified conditions associated with accumulation of the transporter in inward facing conformations. Manipulations that increased the abundance of inward facing states resulted in enhanced steady-state currents. We present a comprehensive kinetic model of the transport cycle, which recapitulates salient features of the recorded currents. This study provides a framework for exploring transporter-associated currents.     

Substrate specificity and ion coupling in the Na+/betaine symporter BetP

BetP is an Na(+)-coupled betaine-specific transporter of the betaine-choline-carnitine (BCC) transporter family involved in the response to hyperosmotic stress. The crystal structure of BetP revealed an overall fold of two inverted structurally related repeats (LeuT-fold) that BetP shares with other sequence-unrelated Na(+)-coupled symporters. Numerous structures of LeuT-fold transporters in distinct conformational states have contributed substantially to our understanding of the alternating access mechanism of transport. Nevertheless, coupling of substrate and co-transported ion fluxes has not been structurally corroborated to the same extent. We converted BetP by a single-point mutation--glycine to aspartate--into an H(+)-coupled choline-specific transporter and solved the crystal structure of this mutant in complex with choline. The structure of BetP-G153D demonstrates a new inward-facing open conformation for BetP. Choline binding to a location close to the second, low-affinity sodium-binding site (Na2) of LeuT-fold transporters is facilitated by the introduced aspartate. Our data confirm the importance of a cation-binding site in BetP, playing a key role in a proposed molecular mechanism of Na(+) and H(+) coupling in BCC transporters.     

A multi-substrate single-file model for ion-coupled transporters.

Ion-coupled transporters are simulated by a model that differs from contemporary alternating-access schemes. Beginning with concepts derived from multi-ion pores, the model assumes that substrates (both inorganic ions and small organic molecules) hop a) between the solutions and binding sites and b) between binding sites within a single-file pore. No two substrates can simultaneously occupy the same site. Rate constants for hopping can be increased both a) when substrates in two sites attract each other into a vacant site between them and b) when substrates in adjacent sites repel each other. Hopping rate constants for charged substrates are also modified by the membrane field. For a three-site model, simulated annealing yields parameters to fit steady-state measurements of flux coupling, transport-associated currents, and charge movements for the GABA transporter GAT1. The model then accounts for some GAT1 kinetic data as well. The model also yields parameters that describe the available data for the rat 5-HT transporter and for the rabbit Na(+)-glucose transporter. The simulations show that coupled fluxes and other aspects of ion transport can be explained by a model that includes local substrate-substrate interactions but no explicit global conformational changes.     

Identification of the Third Na Site and the Sequence of Extracellular Binding Events in the Glutamate Transporter

The transport cycle in the glutamate transporter (GlT) is catalyzed by the cotransport of three Na⁺ ions. However, the positions of only two of these ions (Na1 and Na2 sites) along with the substrate have been captured in the crystal structures reported for both the outward-facing and the inward-facing states of Glt_ph. Characterizing the third ion binding site (Na3) is necessary for structure-function studies attempting to investigate the mechanism of transport in GlTs at an atomic level, particularly for the determination of the sequence of the binding events during the transport cycle. In this study, we report a series of molecular dynamics simulations performed on various bound states of Glt_ph (the apo state, as well as in the presence of Na⁺, the substrate, or both), which have been used to identify a putative Na3 site. The calculated trajectories have been used to determine the water accessibility of potential ion-binding residues in the protein, as a prerequisite for their ion binding. Combined with conformational analysis of the key regions in the protein in different bound states and several additional independent simulations in which a Na⁺ ion was randomly introduced to the interior of the transporter, we have been able to characterize a putative Na3 site and propose a plausible binding sequence for the substrate and the three Na⁺ ions to the transporter during the extracellular half of the transport cycle. The proposed Na3 site is formed by a set of highly conserved residues, namely, Asp³¹², Thr⁹², and Asn³¹⁰, along with a water molecule. Simulation of a fully bound state, including the substrate and the three Na⁺ ions, reveals a stable structure—showing closer agreement to the crystal structure when compared to previous models lacking an ion in the putative Na3 site. The proposed sequence of binding events is in agreement with recent experimental models suggesting that two Na⁺ ions bind before the substrate, and one after that. Our results, however, provide additional information about the sites involved in these binding events.     

Mutational Mapping and Modeling of the Binding Site for (S)-Citalopram in the Human Serotonin Transporter

The serotonin transporter (SERT) regulates extracellular levels of the neurotransmitter serotonin (5-hydroxytryptamine) in the brain by facilitating uptake of released 5-hydroxytryptamine into neuronal cells. SERT is the target for widely used antidepressant drugs, including imipramine, fluoxetine, and (S)-citalopram, which are competitive inhibitors of the transport function. Knowledge of the molecular details of the antidepressant binding sites in SERT has been limited due to lack of structural data on SERT. Here, we present a characterization of the (S)-citalopram binding pocket in human SERT (hSERT) using mutational and computational approaches. Comparative modeling and ligand docking reveal that (S)-citalopram fits into the hSERT substrate binding pocket, where (S)-citalopram can adopt a number of different binding orientations. We find, however, that only one of these binding modes is functionally relevant from studying the effects of 64 point mutations around the putative substrate binding site. The mutational mapping also identify novel hSERT residues that are crucial for (S)-citalopram binding. The model defines the molecular determinants for (S)-citalopram binding to hSERT and demonstrates that the antidepressant binding site overlaps with the substrate binding site.     

4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter

Monoamine transporters terminate synaptic neurotransmission and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening. However, until now, their use has mostly been successful to study the catecholamine transporters but not the serotonin (5HT) transporter. Here, we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP(+)) as reporters for the human serotonin transporter (hSERT) in single cells. The fluorescent substrate 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) exhibits superior fluorescence uptake in hSERT-expressing HEK293 cells than other MPP(+) analogs tested. APP(+) uptake is Na(+)- and Cl(-)-dependent, displaced by 5HT, and inhibited by fluoxetine, suggesting APP(+) specifically monitors hSERT activity. ASP(+), which was previously used to study catecholamine transporters, is 10 times less potent than APP(+) at inhibiting 5HT uptake and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped to -60 mV, APP(+) induced fluoxetine-sensitive hSERT-mediated inward currents, indicating APP(+) is a substrate, whereas ASP(+) induced hSERT-mediated outward currents and counteracted 5HT-induced hSERT currents, indicating ASP(+) possesses activity as an inhibitor. Extra-precise ligand receptor docking of APP(+) and ASP(+) in an hSERT homology model showed both ASP(+) and APP(+) docked favorably within the active region; accordingly, comparable concentrations are required to elicit their opposite electrophysiological responses. We conclude APP(+) is better suited than ASP(+) to study hSERT transport fluorometrically.     

Early intermediates in the transport cycle of the neuronal excitatory amino acid carrier EAAC1

Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EAAC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The molecular nature of these charge movements is poorly understood at present and, therefore, was studied in this report in detail by using the technique of laser-pulse photolysis of caged glutamate providing a 100-micros time resolution. In the inward transport mode, the deactivation of the transient component of the glutamate-induced coupled transport current exhibits two exponential components. Similar results were obtained when restricting EAAC1 to Na(+) translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na(+)-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge movement and of the steady-state EAAC1 properties, we developed a kinetic model that is based on sequential binding of Na(+) and glutamate to their extracellular binding sites on EAAC1 explaining our results. In this model, at least one Na(+) ion and thereafter glutamate rapidly bind to the transporter initiating a slower, electroneutral structural change that makes EAAC1 competent for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, electrogenic translocation reaction to expose the substrate and ion binding sites to the cytoplasm.     

Substrate-dependent gating of anion channels associated with excitatory amino acid transporter 4

EAAT glutamate transporters do not only function as secondary-active glutamate transporters but also as anion channels. EAAT anion channel activity depends on transport substrates. For most isoforms, it is negligible without external Na(+) and increased by external glutamate. We here investigated gating of EAAT4 anion channels with various cations and amino acid substrates using patch clamp experiments on a mammalian cell line. We demonstrate that Li(+) can substitute for Na(+) in supporting substrate-activated anion currents, albeit with changed voltage dependence. Anion currents were recorded in glutamate, aspartate, and cysteine, and distinct time and voltage dependences were observed. For each substrate, gating was different in external Na(+) or Li(+). All features of voltage-dependent and substrate-specific anion channel gating can be described by a simplified nine-state model of the transport cycle in which only amino acid substrate-bound states assume high anion channel open probabilities. The kinetic scheme suggests that the substrate dependence of channel gating is exclusively caused by differences in substrate association and translocation. Moreover, the voltage dependence of anion channel gating arises predominantly from electrogenic cation binding and membrane translocation of the transporter. We conclude that all voltage- and substrate-dependent conformational changes of the EAAT4 anion channel are linked to transitions within the transport cycle.     

The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport

Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na⁺, Cl⁻, and K⁺ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na⁺-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca²⁺ (but not other cations) to functionally replace Na⁺ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca²⁺ and Na⁺ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca²⁺ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na⁺-binding sites.     

Electrogenic ion transport by Na+,K+-ATPase

Experimental data on the ion electrogenic transport by Na+,K+-ATPase available in the literature are analyzed. Special attention is paid to the measurements of unsteady-state electric currents initiated by alternating voltage or rapid introduction of the substrate. In the final part, a physical model of the Na+,K+-ATPase functioning is discussed. According to this model, active transport is carried out by opening and closing of the access channels used for the sodium and potassium exchange between solutions on either side of the membrane. The model explains most of the experimental data, although some details (the channel size, rates of individual transport steps) need further refinement.     

Atomistic models of ion and solute transport by the sodium-dependent secondary active transporters

The recent determination of high-resolution crystal structures of several transporters offers unprecedented insights into the structural mechanisms behind secondary transport. These proteins utilize the facilitated diffusion of the ions down their electrochemical gradients to transport the substrate against its concentration gradient. The structural studies revealed striking similarities in the structural organization of ion and solute binding sites and a well-conserved inverted-repeat topology between proteins from several gene families. In this paper we will overview recent atomistic simulations applied to study the mechanisms of selective binding of ion and substrate in LeuT, Glt, vSGLT and hSERT as well as its consequences for the transporter conformational dynamics. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Pre-steady-state and reverse transport currents in the GABA transporter GAT1

The role of internal substrates in the biophysical properties of the GABA transporter GAT1 has been investigated electrophysiologically in Xenopus oocytes heterologously expressing the cotransporter. Increments in Cl(-) and/or Na(+) concentrations caused by intracellular injections did not produce significant effects on the pre-steady-state currents, while a positive shift of the charge-voltage (Q-V) and decay time constant (τ)-voltage (τ-V) curves, together with a slowing of τ at positive potentials, was observed following treatments producing cytosolic Cl(-) depletion. Activation of the reverse transport mode by injections of GABA caused a reduction in the displaced charge. In the absence of external Cl(-), a stronger reduction in the displaced charge, together with a significant increase in reverse transport current, was observed. Therefore, complementarity between pre-steady-state and transport currents, observed in the forward mode, is preserved in the reverse mode. All these findings can be qualitatively reproduced by a kinetic scheme in which, in the forward mode, the Cl(-) ion is released first, after the inward charge movement, while the two Na(+) ions can be released only after binding of external GABA. In the reverse mode, internal GABA must bind first to the empty transporter, followed by internal Na(+) and Cl(-).     

Distinct conformational states mediate the transport and anion channel properties of the glutamate transporter EAAT-1

Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na(+), 1 H(+), and the counter-transport of 1 K(+) ion. In addition to coupled ion fluxes, glutamate and Na(+) binding to the transporter activates a thermodynamically uncoupled anion conductance through the transporter. In this study, we have distinguished between these two conductance states of the EAAT-1 transporter using a [2-(trimethylammonium)ethyl]methanethiosulfonate-modified V452C mutant transporter. Glutamate binds to the modified mutant transporter and activates the uncoupled anion conductance but is not transported. The selective alteration of the transport function without altering the anion channel function of the V452C mutant transporter suggests that the two functions are generated by distinct conformational states of the transporter.     

GAT1 (GABA:Na+:Cl-) cotransport function. Steady state studies in giant Xenopus oocyte membrane patches.

Neurotransmitter transporters are reported to mediate transmembrane ion movements that are poorly coupled to neurotransmitter transport and to exhibit complex "channel-like" behaviors that challenge the classical "alternating access" transport model. To test alternative models, and to develop an improved model for the Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporter, GAT1, we expressed GAT1 in Xenopus oocytes and analyzed its function in detail in giant membrane patches. We detected no Na+- or Cl--dependent currents in the absence of GABA, nor did we detect activating effects of substrates added to the trans side. Outward GAT1 current ("reverse" transport mode) requires the presence of all three substrates on the cytoplasmic side. Inward GAT1 current ("forward" transport mode) can be partially activated by GABA and Na+ on the extracellular (pipette) side in the nominal absence of Cl-. With all three substrates on both membrane sides, reversal potentials defined with specific GAT1 inhibitors are consistent with the proposed stoichiometry of 1GABA:2Na+:1Cl-. As predicted for the "alternating access" model, addition of a substrate to the trans side (120 mM extracellular Na+) decreases the half-maximal concentration for activation of current by a substrate on the cis side (cytoplasmic GABA). In the presence of extracellular Na+, the half-maximal cytoplasmic GABA concentration is increased by decreasing cytoplasmic Cl-. In the absence of extracellular Na+, half-maximal cytoplasmic substrate concentrations (8 mM Cl-, 2 mM GABA, 60 mM Na+) do not change when cosubstrate concentrations are reduced, with the exception that reducing cytoplasmic Cl- increases the half-maximal cytoplasmic Na+ concentration. The forward GAT1 current (i.e., inward current with all extracellular substrates present) is inhibited monotonically by cytoplasmic Cl- (Ki, 8 mM); cytoplasmic Na+ and cytoplasmic GABA are without effect in the absence of cytoplasmic Cl-. In the absence of extracellular Na+, current-voltage relations for reverse transport current (i.e., outward current with all cytoplasmic substrates present) can be approximated by shallow exponential functions whose slopes are consistent with rate-limiting steps moving 0.15-0.3 equivalent charges. The slopes of current-voltage relations change only little when current is reduced four- to eightfold by lowering each cosubstrate concentration; they increase twofold upon addition of 100 mM Na+ to the extracellular (pipette) side.     

Simulations of the alternating access mechanism of the sodium symporter Mhp1

Sodium coupled cotransporters of the five-helix inverted repeat (5HIR) superfamily use an alternating access mechanism to transport a myriad of small molecules across the cell membrane. One of the primary steps in this mechanism is the conformational transition from a state poised to bind extracellular substrates to a state that is competent to deliver substrate to the cytoplasm. Here, we construct a coarse-grained model of the 5HIR benzylhydantoin transporter Mhp1 that incorporates experimental structures of the outward- and inward-open states to investigate the mechanism of this conformational change. Using the weighted ensemble path-sampling method, we rigorously sample the outward- to inward-facing transition path ensemble. The transition path ensemble reveals a heterogeneous set of pathways connecting the two states and identifies two modes of transport: one consistent with a strict alternating access mechanism and another where decoupling of the inner and outer gates causes the transient formation of a continuous permeation pathway through the transporter. We also show that the conformational switch between the outward- and inward-open states results from rigid body motions of the hash motif relative to the substrate bundle, supporting the rocking bundle hypothesis. Finally, our methodology provides the groundwork for more chemically detailed investigations of the alternating mechanism.     

GAT1 (GABA:Na+:Cl-) cotransport function. Database reconstruction with an alternating access model.

We have developed an alternating access transport model that accounts well for GAT1 (GABA:Na+:Cl-) cotransport function in Xenopus oocyte membranes. To do so, many alternative models were fitted to a database on GAT1 function, and discrepancies were analyzed. The model assumes that GAT1 exists predominantly in two states, Ein and E(out). In the Ein state, one chloride and two sodium ions can bind sequentially from the cytoplasmic side. In the Eout state, one sodium ion is occluded within the transporter, and one chloride, one sodium, and one gamma-aminobutyric acid (GABA) molecule can bind from the extracellular side. When Ein sites are empty, a transition to the Eout state opens binding sites to the outside and occludes one extracellular sodium ion. This conformational change is the major electrogenic GAT1 reaction, and it rate-limits forward transport (i.e., GABA uptake) at 0 mV. From the Eout state, one GABA can be translocated with one sodium ion to the cytoplasmic side, thereby forming the *Ein state. Thereafter, an extracellular chloride ion can be translocated and the occluded sodium ion released to the cytoplasm, which returns the transporter to the Ein state. GABA-GABA exchange can occur in the absence of extracellular chloride, but a chloride ion must be transported to complete a forward transport cycle. In the reverse transport cycle, one cytoplasmic chloride ion binds first to the Ein state, followed by two sodium ions. One chloride ion and one sodium ion are occluded together, and thereafter the second sodium ion and GABA are occluded and translocated. The weak voltage dependence of these reactions determines the slopes of outward current-voltage relations. Experimental results that are simulated accurately include (a) all current-voltage relations, (b) all substrate dependencies described to date, (c) cis-cis and cis-trans substrate interactions, (d) charge movements in the absence of transport current, (e) dependencies of charge movement kinetics on substrate concentrations, (f) pre-steady state current transients in the presence of substrates, (g) substrate-induced capacitance changes, (h) GABA-GABA exchange, and (i) the existence of inward transport current and GABA-GABA exchange in the nominal absence of extracellular chloride.     

Transported Substrate Determines Exchange Rate in the Multidrug Resistance Transporter EmrE

EmrE, a small multidrug resistance transporter, serves as an ideal model to study coupling between multidrug recognition and protein function. EmrE has a single small binding pocket that must accommodate the full range of diverse substrates recognized by this transporter. We have studied a series of tetrahedral compounds, as well as several planar substrates, to examine multidrug recognition and transport by EmrE. Here we show that even within this limited series, the rate of interconversion between the inward- and outward-facing states of EmrE varies over 3 orders of magnitude. Thus, the identity of the bound substrate controls the rate of this critical step in the transport process. The binding affinity also varies over a similar range and is correlated with substrate hydrophobicity within the tetrahedral substrate series. Substrate identity influences both the ground-state and transition-state energies for the conformational exchange process, highlighting the coupling between substrate binding and transport required for alternating access antiport.     

A high affinity fungal nitrate carrier with two transport mechanisms

We have expressed the CRNA high affinity nitrate transporter from Emericella (Aspergillus) nidulans in Xenopus oocytes and used electrophysiology to study its properties. This method was used because there are no convenient radiolabeled substrates for the transporter. Oocytes injected with crnA mRNA showed nitrate-, nitrite-, and chlorite-dependent currents. Although the gene was originally identified by chlorate selection there was no evidence for transport of this anion. The gene selection is explained by the high affinity of the transporter for chlorite, and the fact that this ion contaminates solutions of chlorate. The pH-dependence of the anion-elicited currents was consistent with H(+)-coupled mechanism of transport. At any given voltage, currents showed hyperbolic kinetics with respect to extracellular H(+), and these data could be fitted with a Michaelis-Menten relationship. But this equation did not adequately describe transport of the anion substrates. At higher concentrations of the anion substrates and more negative membrane voltages, the currents were decreased, but this effect was independent of changes in external pH. These more complicated kinetics could be fit by an equation containing two Michaelis-Menten terms. The substrate inhibition of the currents could be explained by a transport reaction cycle that included two routes for the transfer of nitrate across the membrane, one on the empty carrier and the other proton coupled. The model predicts that the substrate inhibition of transporter current depends on the cytosolic nitrate concentration. This is the first time a high affinity nitrate transport activity has been characterized in a heterologous system and the measurements show how the properties of the CRNA transporter are modified by changes in the membrane potential, external pH, and nitrate concentration. The physiological significance of these observations is discussed.