Different functional roles of arginine residues 39 and 61 and tyrosine residue 98 in transport and channel mode of the glutamate transporter EAAC1

The excitatory amino acid transporter EAAC1 is an electrogenic Na+ - and K+ -gradient-driven transporter. In addition, the transporter mediates in the presence of Na+ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the transporter family. Also the conserved tyrosine residue Y98 could be important for Cl- conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent Km values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent Km value for glutamate, and with strong reduction of the selectivity ration between NO3- and Cl- of the conductance mode.     

Water and urea permeation pathways of the human excitatory amino acid transporter EAAT1

Glutamate transport is coupled to the co-transport of 3 Na(+) and 1 H(+) followed by the counter-transport of 1 K(+). In addition, glutamate and Na(+) binding to glutamate transporters generates an uncoupled anion conductance. The human glial glutamate transporter EAAT1 (excitatory amino acid transporter 1) also allows significant passive and active water transport, which suggests that water permeation through glutamate transporters may play an important role in glial cell homoeostasis. Urea also permeates EAAT1 and has been used to characterize the permeation properties of the transporter. We have previously identified a series of mutations that differentially affect either the glutamate transport process or the substrate-activated channel function of EAAT1. The water and urea permeation properties of wild-type EAAT1 and two mutant transporters were measured to identify which permeation pathway facilitates the movement of these molecules. We demonstrate that there is a significant rate of L-glutamate-stimulated passive and active water transport. Both the passive and active L-glutamate-stimulated water transport is most closely associated with the glutamate transport process. In contrast, L-glutamate-stimulated [(14)C]urea permeation is associated with the anion channel of the transporter. However, there is also likely to be a transporter-specific, but glutamate independent, flux of water via the anion channel.     

Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.     

A Mutation in Transmembrane Domain 7 (TM7) of Excitatory Amino Acid Transporters Disrupts the Substrate-dependent Gating of the Intrinsic Anion Conductance and Drives the Channel into a Constitutively Open State

In the mammalian central nervous system, excitatory amino acid transporters (EAATs) are responsible for the clearance of glutamate after synaptic release. This energetically demanding activity is crucial for precise neuronal communication and for maintaining extracellular glutamate concentrations below neurotoxic levels. In addition to their ability to recapture glutamate from the extracellular space, EAATs exhibit a sodium- and glutamate-gated anion conductance. Here we show that substitution of a conserved positively charged residue (Arg-388, hEAAT1) in transmembrane domain 7 with a negatively charged amino acid eliminates the ability of glutamate to further activate the anion conductance. When expressed in oocytes, R388D or R388E mutants show large anion currents that display no further increase in amplitude after application of saturating concentrations of Na⁺ and glutamate. They also show a substantially reduced transport activity. The mutant transporters appear to exist preferentially in a sodium- and glutamate-independent constitutive open channel state that rarely transitions to complete the transport cycle. In addition, the accessibility of cytoplasmic residues to membrane-permeant modifying reagents supports the idea that this substrate-independent open state correlates with an intermediate outward facing conformation of the transporter. Our data provide additional insights into the mechanism by which substrates gate the anion conductance in EAATs and suggest that in EAAT1, Arg-388 is a critical element for the structural coupling between the substrate translocation and the gating mechanisms of the EAAT-associated anion channel.     

The chloride permeation pathway of a glutamate transporter and its proximity to the glutamate translocation pathway

Excitatory amino acid transporters (EAATs) regulate glutamate concentrations in the brain to maintain normal excitatory synaptic transmission. A widely accepted view of transporters is that they consist of a pore with alternating access to the intracellular and extracellular solutions, which serves to couple ion movement to the movement of substrate. However, recent observations that EAATs, and also a number of other neurotransmitter transporters, can also function as ligand-gated chloride channels have blurred the distinctions between transporters and ion channels. Here we show that mutations in the second transmembrane domain (TM2) of EAAT1 alter anion permeation properties without affecting glutamate transport and that a number of TM2 residues are accessible to the external aqueous solution. Furthermore, we demonstrate that the extracellular edge of TM2 is in close proximity to a membrane-associated domain that influences glutamate transport. This study will provide the foundation for beginning to understand how transporters can function as both transporters and ion channels.     

Zn(2+) inhibits the anion conductance of the glutamate transporter EEAT4

Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na(+) ions and 1 H(+) and the counter-transport of 1 K(+) ion, which ensures that extracellular glutamate concentrations are maintained in the submicromolar range. In addition to the coupled ion fluxes, glutamate transport activates an uncoupled anion conductance that does not influence the rate or direction of transport but may have the capacity to influence the excitability of the cell. Free Zn(2+) ions are often co-localized with glutamate in the central nervous system and have the capacity to modulate the dynamics of excitatory neurotransmission. In this study we demonstrate that Zn(2+) ions inhibit the uncoupled anion conductance and also reduce the affinity of L-aspartate for EAAT4. The molecular basis for this effect was investigated using site-directed mutagenesis. Two histidine residues in the extracellular loop between transmembrane domains three and four of EAAT4 appear to confer Zn(2+) inhibition of the anion conductance.     

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.     

Molecular Determinants for Functional Differences between Alanine-Serine-Cysteine Transporter 1 and Other Glutamate Transporter Family Members

The ASCTs (alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter Glt_Ph. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and Glt_Ph. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and Glt_Ph, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and Glt_Ph. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly l-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of Glt_Ph, with preference for l-aspartate over l-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH 5.5; however, this was reversed by the additional mutation A382T. We propose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family.     

Functional Properties of the Retinal Glutamate Transporters GLT-1c and EAAT5

In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as “excitatory amino acid transporters (EAATs).” Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na⁺ and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na⁺ and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.     

Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels

Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels.     

Neutralization of the aspartic acid residue Asp-367, but not Asp-454, inhibits binding of Na+ to the glutamate-free form and cycling of the glutamate transporter EAAC1

Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na(+) ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, Asp-367 and Asp-454, in Na(+) cotransport. To test the effect of charge neutralization mutations in these positions on Na(+) binding to the glutamate-free transporter, we recorded the Na(+)-induced anion leak current to determine the K(m) of EAAC1 for Na(+). For EAAC1(WT), this K(m) was determined as 120 mm. When the negative charge of Asp-367 was neutralized by mutagenesis to asparagine, Na(+) activated the anion leak current with a K(m) of about 2 m, indicating dramatically impaired Na(+) binding to the mutant transporter. In contrast, the Na(+) affinity of EAAC1(D454N) was virtually unchanged compared with the wild type transporter (K(m) = 90 mm). The reduced occupancy of the Na(+) binding site of EAAC1(D367N) resulted in a dramatic reduction in glutamate affinity (K(m) = 3.6 mm, 140 mm [Na(+)]), which could be partially overcome by increasing extracellular [Na(+)]. In addition to impairing Na(+) binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which Asp-367, but not Asp-454, is involved in coordinating the bound Na(+) in the glutamate-free transporter form.     

The fourth transmembrane domain of the Helicobacter pylori Na+/H+ antiporter NhaA faces a water-filled channel required for ion transport

Cysteine-scanning mutagenesis was performed from Ser-130 to Leu-160 in the fourth transmembrane domain (TM4) of the Na+/H+ antiporter NhaA from Helicobacter pylori to determine the topology of each residue and to identify functionally important residues. All of the mutants were based on cysteine-less NhaA (Cys-less NhaA), which functions very similarly to the wild-type protein, and were expressed at a level similar to Cys-less NhaA. Discontinuity of [14C]N-ethylmaleimide (NEM)-reactive residues suggested that TM4 comprises residues Gly-135 to Val-156. Even within TM4, NEM reactivity was high for I136C, D141C to A143C, L146C, M150C, and G153C to R155C. These residues are thought to be located on one side of the -helical structure of TM4 and to face a putative water-filled channel. Pretreatment of intact cells with membrane-impermeable maleimide did not inhibit [14C]NEM binding to the NEM-reactive residues within TM4, suggesting that the putative channel opens toward the cytoplasm. NEM reactivity of the A143C mutant was significantly inhibited by Li+. The T140C and D141C mutants showed lower affinity for Na+ and Li+ as transport substrates, but their maximal antiporter velocities (Vmax) were relatively unaffected. Whereas the I142C and F144C mutants completely lost their Li+/H+ antiporter activity, I142C had a lower Vmax for the Na+/H+ antiporter. F144C exhibited a markedly lower Vmax and a partially reduced affinity for Na+. These results suggest that Thr-140, Asp-141, and Phe-144 are located in the end portion of a putative water-filled channel and may provide the binding site for Na+, Li+, and/or H+. Furthermore, residues Ile-142 to Phe-144 may be important for the conformational change that accompanies ion transport in NhaA.     

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.     

Neutralizing aspartate 83 modifies substrate translocation of excitatory amino acid transporter 3 (EAAT3) glutamate transporters

Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission by removing glutamate from the synaptic cleft into neuronal and glial cells. EAATs are not only secondary active glutamate transporters but also function as anion channels. Gating of EAAT anion channels is tightly coupled to transitions within the glutamate uptake cycle, resulting in Na(+)- and glutamate-dependent anion currents. A point mutation neutralizing a conserved aspartic acid within the intracellular loop close to the end of transmembrane domain 2 was recently shown to modify the substrate dependence of EAAT anion currents. To distinguish whether this mutation affects transitions within the uptake cycle or directly modifies the opening/closing of the anion channel, we used voltage clamp fluorometry. Using three different sites for fluorophore attachment, V120C, M205C, and A430C, we observed time-, voltage-, and substrate-dependent alterations of EAAT3 fluorescence intensities. The voltage and substrate dependence of fluorescence intensities can be described by a 15-state model of the transport cycle in which several states are connected to branching anion channel states. D83A-mediated changes of fluorescence intensities, anion currents, and secondary active transport can be explained by exclusive modifications of substrate translocation rates. In contrast, sole modification of anion channel opening and closing is insufficient to account for all experimental data. We conclude that D83A has direct effects on the glutamate transport cycle and that these effects result in changed anion channel function.     

Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1

Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na(+) ions, one H(+) and the counter-transport of one K(+) ion. Transport by an archaeal homologue of the human glutamate transporters, Glt(Ph), whose three dimensional structure is known is also coupled to three Na(+) ions but only two Na(+) ion binding sites have been observed in the crystal structure of Glt(Ph). In order to fully utilize the Glt(Ph) structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of Glt(Ph) and accurately determine the number and location of Na(+) ions coupled to transport. Several sites have been proposed for the binding of a third Na(+) ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for Glt(Ph) and reveal a new site for the third Na(+) ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in Glt(Ph), and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na(+) compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na(+) ion in Glt(Ph) and EAAT1.     

Charge conversion enables quantification of the proximity between a normally-neutral mu-conotoxin (GIIIA) site and the Na+ channel pore

mu-Conotoxin (mu-CTX) inhibits Na+ flux by obstructing the Na+ channel pore. Previous studies of mu-CTX have focused only on charged toxin residues, ignoring the neutral sites. Here we investigated the proximity between the C-terminal neutral alanine (A22) of mu-CTX and the Na+ channel pore by replacing it with the negatively charged glutamate. The analog A22E and wild-type (WT) mu-CTX exhibited identical nuclear magnetic resonance spectra except at the site of replacement, verifying that they have identical backbone structures. A22E significantly reduced mu-CTX affinity for WT mu1 Na+ channels (90-fold), as if the inserted glutamate repels the anionic pore receptor. We then looked for the interacting partner(s) of residue 22 by determining the potency of block of Y401K, Y401A, E758Q, D762K, D762A, E765K, E765A and D1241K channels by WT mu-CTX and A22E, followed by mutant cycle analysis to assess their individual couplings. Our results show that A22E interacts strongly with E765K from domain II (DII) (deltadeltaG=2.2 +/- 0.1 vs. <1 kcal/mol for others). We conclude that mu-CTX residue 22 closely associates with the DII pore in the toxin-bound channel complex. The approach taken may be further exploited to study the proximity of other neutral toxin residues with the Na+ channel pore.     

Second transmembrane domain of human uncoupling protein 2 is essential for its anion channel formation

Uncoupling proteins (UCP) are known to transport anions, such as Cl-, in addition to H+ transport. Although H+ transport by UCP is clearly involved in thermogenesis, the mechanism of its anion transport is not clearly understood. In this study, we examined the anion channel characteristics of the six individual helical transmembrane (TM) domains of the human UCP2. The second TM domain peptide (TM2) forms multi-state channels by assemblies of conductive oligomers. Furthermore, the TM2 exhibited voltage-dependent anion channels with properties comparable to those of UCP1 chloride channel. However, the other five TM peptides did not form UCP1-like channels. Moreover, an analog of TM2 in which two Arg residues were substituted by Ala residues did not form stable channels, implying the significance of Arg residues for anion transport. These results suggest that the anion channel structure of UCP2 protein is oligomeric and the second TM domain is essential for the voltage-dependence of this anion channel.     

Mutating a Conserved Proline Residue within the Trimerization Domain Modifies Na+ Binding to Excitatory Amino Acid Transporters and Associated Conformational Changes

Excitatory amino acid transporters (EAATs) are crucial for glutamate homeostasis in the mammalian central nervous system. They are not only secondary active glutamate transporters but also function as anion channels, and different EAATs vary considerably in glutamate transport rates and associated anion current amplitudes. A naturally occurring mutation, which was identified in a patient with episodic ataxia type 6 and that predicts the substitution of a highly conserved proline at position 290 by arginine (P290R), was recently shown to reduce glutamate uptake and to increase anion conduction by hEAAT1. We here used voltage clamp fluorometry to define how the homologous P259R mutation modifies the functional properties of hEAAT3. P259R inverts the voltage dependence, changes the sodium dependence, and alters the time dependence of hEAAT3 fluorescence signals. Kinetic analysis of fluorescence signals indicate that P259R decelerates a conformational change associated with sodium binding to the glutamate-free mutant transporters. This alteration in the glutamate uptake cycle accounts for the experimentally observed changes in glutamate transport and anion conduction by P259R hEAAT3.     

Interactions of alkali cations with glutamate transporters

The transport of glutamate is coupled to the co-transport of three Na+ ions and the countertransport of one K+ ion. In addition to this carrier-type exchange behaviour, glutamate transporters also behave as chloride channels. The chloride channel activity is strongly influenced by the cations that are involved in coupled flux, making glutamate transporters representative of the ambiguous interface between carriers and channels. In this paper, we review the interaction of alkali cations with glutamate transporters in terms of these diverse functions. We also present a model derived from electrostatic mapping of the predicted cation-binding sites in the X-ray crystal structure of the Pyrococcus horikoshii transporter GltPh and in its human glutamate transporter homologue EAAT3. Two predicted Na+-binding sites were found to overlap precisely with the Tl+ densities observed in the aspartate-bound complex. A novel third site predicted to favourably bind Na+ (but not Tl+) is formed by interaction with the substrate and the occluding HP2 loop. A fourth predicted site in the apo state exhibits selectivity for K+ over both Na+ and Tl+. Notably, this K+ site partially overlaps the glutamate-binding site, and their binding is mutually exclusive. These results are consistent with kinetic and structural data and suggest a plausible mechanism for the flux coupling of glutamate with Na+ and K+ ions.     

Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3

Organic anion transporters (OATs) and organic cation transporters (OCTs) mediate the flux of xenobiotics across the plasma membranes of epithelia. Substrates of OATs generally carry negative charge(s) whereas substrates of OCTs are cations. The goal of this study was to determine the domains and amino acid residues essential for recognition and transport of organic anions by the rat organic anion transporter, rOAT3. An rOAT3/rOCT1 chimera containing transmembrane domains 1-5 of rOAT3 and 6-12 of rOCT1 retained the specificity of rOCT1, suggesting that residues involved in substrate recognition reside within the carboxyl-terminal half of these transporters. Mutagenesis of a conserved basic amino acid residue, arginine 454 to aspartic acid (R454D), revealed that this amino acid is required for organic anion transport. The uptakes of p-aminohippurate (PAH), estrone sulfate, and ochratoxin A were approximately 10-, approximately 48-, and approximately 32-fold enhanced in oocytes expressing rOAT3 and were only approximately 2-, approximately 6-, and approximately 5-fold enhanced for R454D. Similarly, mutagenesis of the conserved lysine 370 to alanine (K370A) suggested that K370 is important for organic anion transport. Interestingly, the charge specificity of the double mutant, R454DK370A, was reversed in comparison to rOAT3-R454DK370A preferentially transported the organic cation, MPP(+), in comparison to PAH (MPP(+) uptake/PAH uptake = 3.21 for the double mutant vs 0.037 for rOAT3). These data indicate that arginine 454 and lysine 370 are essential for the anion specificity of rOAT3. The studies provide the first insights into the molecular determinants that are critical for recognition and translocation of organic anions by a member of the organic anion transporter family.