©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Electrogenic Properties of the Epithelial and Neuronal High Affinity Glutamate Transporter (*)

Yoshikatsu Kanai (§) , Stephan Nussberger , Michael F. Romero (1), Walter F. Boron (1), Steven C. Hebert , Matthias A. Hediger (¶)

From the (1)Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 Department of Cellular and Molecular Physiology, Yale University, School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Active ion-coupled glutamate transport is of critical importance for excitatory synaptic transmission, normal cellular function, and epithelial amino acid metabolism. We previously reported the cloning of the rabbit intestinal high affinity glutamate transporter EAAC1 (Kanai, Y., and Hediger, M. A.(1992) Nature 360, 467-471), which [Medline] is expressed in numerous tissues including intestine, kidney, liver, heart, and brain. Here, we report a detailed stoichiometric and kinetic analysis of EAAC1 expressed in Xenopus laevis oocytes. Uptake studies of Na and [C]glutamate, in combination with measurements of intracellular pH with pH microelectrodes gave a glutamate to charge ratio of 1:1, a glutamate to Na ratio of 1:2, and a OH/H to charge ratio of 1:1. Since transport is K dependent it can be concluded that EAAC1-mediated glutamate transport is coupled to the cotransport of 2 Na ions, the countertransport of one K ion and either the countertransport of one OH ion or the cotransport of 1 H ion. We further demonstrate that under conditions where the electrochemical gradients for these ions are disrupted, EAAC1 runs in reverse, a transport mode which is of pathologic importance. Na uptake studies revealed that there is a low level of Na uptake in the absence of extracellular glutamate which appears to be analogous to the Na leak observed for the intestinal Na/glucose cotransporter SGLT1. In voltage clamp studies, reducing extracellular Na from 100 to 10 mM strongly increased K and decreased I. The data indicate that Na binding at the extracellular transporter surface becomes rate-limiting. Studies addressing the cooperativity of the substrate-binding sites indicate that there are two distinct Na-binding sites with different affinities and that Na binding is modulated by extracellular glutamate. A hypothetical ordered kinetic transport model for EAAC1 is discussed.


INTRODUCTION

For glutamate to fulfill its diverse functions, the existence of active cellular glutamate uptake systems with a high accumulative power is of critical importance. The cDNAs encoding four different mammalian high affinity glutamate transporter isoforms have been recently cloned and characterized. These are the neuronal and epithelial glutamate transporter EAAC1 (EAAT3)(1, 2) , the glial glutamate transporters GLT-1 (EAAT2) (3, 4) and GLAST (EAAT1)(4, 5) , and the cerebellar glutamate transporter EAAT4(6) . These transporters exhibit 39-55% sequence identities with each other.

Studies of glutamate transport in salamander retina glial cells suggest that transport is electrogenic and coupled to the cotransport of two Na ions and the countertransport of one K and one OH ion(7, 8) . The coupling pattern has not yet been conclusively investigated for the recombinant high affinity glutamate transporters. Knowledge of stoichiometry of cloned glutamate transporters, however, is of critical importance to determine their structure-function relationships and to understand their physiological and pathophysiological roles.

Recent electrophysiological studies of electrogenic transporters provided important clues to how uphill solute transport is coupled to downhill electrochemical ion gradients. Experiments using the voltage jump paradigm to study the Na/glucose cotransporter (SGLT1) (9) and the Na/Cl/GABA (10) transporter demonstrated current relaxation in response to voltage jumps similar to gating currents observed in voltage-gated ion channels. These currents were interpreted to reflect charge movement within the membrane electric field that is associated with conformational changes of the transporter molecule. It is likely that the identification of charged residues in these transporters that undergo transient movements will provide important information on the structure-function relationships of ion-coupled solute transporters.

In the central nervous system, the coupling stoichiometry of EAAC1 also has pathologic implications. The reduced energy supply that occurs during ischemia after a stroke or during anoxia is known to lead to reduced ATP levels and reduced function of Na,K-ATPase and to result in rundown of electrochemical ion gradients(11) . The disrupted ion gradients are thought to cause high affinity glutamate transporters to run in reverse resulting in a non-vesicular release of glutamate into the synaptic cleft. Since extracellular glutamate is highly toxic to neurons, reversed glutamate transport likely results in neuronal death as a result of these pathologic conditions.

In the present study we investigated the electrogenic characteristics of EAAC1 using the voltage jump technique. We determined the coupling of EAAC1-mediated transport to inorganic ions, and we demonstrate that EAAC1 runs in reverse if we mimic the ischemic condition.


MATERIALS AND METHODS

Xenopus Oocyte Expression of EAAC1

cRNAs were in vitro transcribed from cDNAs in pSPORT1 using T7 RNA polymerase (1). Xenopus oocyte expression studies were performed as described previously (12, 13) using collagenase-treated and manually defolliculated oocytes injected with 50 nl of water or cRNA (25 ng/oocyte). Oocytes were incubated in modified Barth's medium (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO), 0.41 mM CaCl, 0.82 mM MgSO, 2.4 mM NaHCO, 10 mM HEPES, pH 7.4) supplemented with gentamicin (50 µg/ml). Oocytes were used 3-6 days after injection for electrophysiological analyses and flux studies.

Determination of the Glutamate to Charge Stoichiometry

The glutamate to charge stoichiometry was determined by comparing [C]glutamate uptake and glutamate-induced inward current as described (14, 15) with some modifications. Five days after injection, [C]L-glutamate uptake (20 µM) by EAAC1 cRNA-injected oocytes and water-injected control oocytes were measured in standard uptake solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl, 1 mM MgCl, 10 mM HEPES, 5 mM Tris, pH 7.4) for 1, 2, 3, 4, and 5 min. Immediately after completion of the uptake measurements, two microelectrode voltage clamp experiments (2) were performed using the same batch of cRNA- or water-injected oocytes. The oocytes were bathed in the standard uptake solution, and the membrane potential was monitored after impaling oocytes. The bath medium was then changed to the uptake solution containing 20 µM of glutamate, and the membrane potential was measured. After washing with uptake solution without glutamate, each oocyte was clamped at this membrane potential. Inward currents evoked by bath-applied 20 µM glutamate at the above determined holding potentials were recorded, and the values were converted to the rate of charge flux using Faraday's constant (9.65 10 C/mol).

Analysis of Na Coupling Based on the Hill Equation

L-Glutamate evoked currents were measured in uptake media containing n mM NaCl (where n was varied from 0 to 100 mM), 100 minus n mM choline chloride, 2 mM KCl, 1 mM CaCl, 1 mM MgCl, 10 mM HEPES, 5 mM Tris, pH 7.4. The currents were fitted by the Hill equation, and the Hill coefficients were calculated for each glutamate concentration.

Determination of the Na to Glutamate Coupling Ratio

To determine the Na to glutamate stoichiometry, the uptakes of Na and [C]L-glutamate were compared in the same batch of EAAC1 cRNA-injected oocytes. Groups of 8-10 oocytes were preincubated for 30 min in 1 ml of uptake medium containing 40 mM NaCl, 60 mM choline-chloride, 2 mM KCl, 1 mM CaCl, 1 mM MgCl, 1 mM ouabain, 0.1 mM amiloride, 0.1 mM bumetanide, 10 mM HEPES, 5 mM Tris, pH 7.4. Glutamate uptake was measured in the uptake solution containing 2 µCi of [C]L-glutamate and unlabeled L-glutamate to make up the final concentration of 1.0 and 0.2 mM. After 20 min of incubation, oocytes were washed with ice-cold Na-free uptake medium in which NaCl was replaced with choline-Cl. In separate experiments Na uptake was measured for 20 min in the uptake medium containing 20 µCi of Na and 0.2 or 1.0 mM of non-labeled L-glutamate.

Measurement of Intracellular pH Using pH Microelectrodes

Intracellular pH (pH) changes associated with EAAC1-mediated glutamate transport in oocytes were measured by monitoring pH of oocytes using a pH-sensitive microelectrode filled with a hydrogen-selective ionophore(2, 16, 17) . Oocytes were studied 3-7 days after injection of EAAC1 cRNA or water. Briefly, V electrodes were pulled from 2 mm diameter fiber-filled borosilicate capillaries (Warner Instruments). These had resistances of 1-10 M when back-filled with 3 M KCl. The pH electrodes were made from similar pipettes but were silanized by adding 20 µl of tri-n-butyl-chlorosilane to an enclosed container at 200 °C. Pipettes were cooled under vacuum, their tips were filled with Fluka hydrogen ionophore I-mixture B, and backfilled with 0.04 M KHPO, 0.023 M NaOH, 0.015 M NaCl, pH 7.0. The arrangement of the electronics was as described previously (16, 17). Voltage due to pH was obtained by electronically subtracting the signals from the pH and voltage electrodes. V was obtained by subtracting the signals from the voltage electrode and the external reference electrode. Electrometer outputs were directed to an 80386-based computer via a 12-bit analog-to-digital converter, and also to a strip chart recorder. Rates of pH change (dpH/dt) were determined by fitting a line to the relevant portion of the pHversus time record. We determined the portion of dpH/dt due to the EAAC1 transporter by subtracting the dpH/dt obtained during a control period from the dpH/dt obtained immediately thereafter in the presence of L-glutamate. In these experiments, the chamber was perfused with ND96, pH 7.40, followed by 100 µML-glutamate in ND96. We obtained the flux (expressed as picomole cm s) due to the transporter by multiplying the difference dpH/dt by the intracellular buffering power and the surface-to-volume ratio of the oocyte. was determined by measuring the dpH due to the addition of 1.5% CO, 10 mM HCO, ND96 at pH 7.4 and had a value of 13 ± 4 mM/pH unit (n = 18)(18) . The surface to volume ratio was obtained from the oocyte diameter, assuming the cell is a sphere and the oocyte volume is 0.25 µl(19) .

K Dependence

The K dependence of the glutamate-evoked current was determined as described previously (1) over a range of K concentrations of 0-50 mM under voltage clamp condition at holding potentials of -30 and -60 mV. Currents induced by application of 50 µM glutamate were measured in EAAC1 cRNA-injected oocytes.

Reversed Glutamate Transport

Oocytes expressing EAAC1 were impaled in standard ND96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl, 1 mM MgCl, 5 mM HEPES, pH 7.4) and voltage-clamped. After stabilization of the current at -60 mV, oocytes were bathed in high K/high glutamate/zero Na medium (98 mM KCl, 1.8 mM CaCl, 1 mM MgCl, 10 mML-glutamate, 5 mM HEPES, pH 7.4). The bath solution was then replaced by high K medium without glutamate to observe the outward current which is due to reversed glutamate transport. After 45 s, the bathing solution was replaced by high K/high glutamate/zero Na medium to terminate the reversed glutamate transport. To determine the voltage dependence of reversed transport, the outward currents were measured at holding potentials between -100 mV and +30 mV. The reversed transport of EAAC1 was also examined in voltage jump experiments (see below). Oocytes expressing EAAC1 were clamped at -50 mV, and voltage steps were applied as described below while bathed either in the high K, 10 mM glutamate, 0 mM Na medium or in the high K/0 mM glutamate, 0 mM Na medium.

Voltage Jump Studies

Oocytes were subjected to two-microelectrode voltage clamping and command potentials were applied and controlled by an IBM compatible computer via the software CLAMPEX from pCLAMP (version 5.5, Axon Instruments)(2) . The oocyte membrane was held at -50 mV and pulsed to the test potential for 76 ms followed by a 1-s interpulse interval at the holding potential of -50 mV before next pulse. Currents were low pass-filtered at 50 kHz, digitized at 200 µs (512 samples), and saved on computer. Steady-state currents during the voltage jump were obtained by averaging the current during the final 4 ms of the 76 ms jump (average of 10 samples). Steady-state currents were measured for each test potential in the presence and absence of L-glutamate, and the glutamate-induced currents were taken as the difference between these currents. The concentration dependence of L-glutamate evoked currents at any given membrane potential and at different extracellular Na concentrations (Na)()were fitted by the Michaelis-Menten equation. Apparent K and I values were calculated and plotted as a function of membrane potential.


RESULTS

Stoichiometry

The glutamate to charge flux ratio was determined by comparing the initial rate of [C]glutamate uptake and the net charge flux calculated from the glutamate-induced inward current. We have used a similar approach to determine the Na- to glucose-coupling ratio of the high and low affinity Na/glucose cotransporters SGLT1 and SGLT2(14) , and the H- to peptide-coupling ratio of the oligopeptide transporter PepT1(15) .

The uptake of [C]glutamate (20 µM) was linear over the first 5 min (Fig. 1a). The slope of this curve gave the glutamate influx, which was 293 ± 27 fmol/oocyte/s. The charge flux computed from the amplitude of the inward currents induced by application of 20 µM glutamate to oocytes injected with EAAC1 cRNA, and Faraday's constant, was 350 ± 34 fmol/oocyte/s. The holding potential used to determine the inward current for each oocyte corresponded to the steady-state potential in the presence 20 µM glutamate in the bath and was -38 mV. This gave a glutamate to charge flux ratio of 1: 1.19 (Fig. 1b). Transport of glutamate is therefore associated with the translocation of one net positive charge.


Figure 1: Glutamate to charge stoichiometry. The initial rate of the [C]glutamate uptake (a) and the net charge flux calculated from the glutamate-induced inward current are directly compared (b). The glutamate-induced inward current was converted to the charge flux using Faraday's constant. The holding potential was set at the resting potential measured for each oocyte in the presence of 20 µM bath-applied glutamate. The [C]glutamate uptake studies and the electrophysiological measurements were performed on the same batch of EAAC1 cRNA-injected oocytes. The glutamate to charge flux ration was 1:1.19 (b). Glutamate translocation is therefore associated with the translocation of one positive charge.



The Na- to glutamate-coupling ratio was initially studied by measuring the Na dependence of glutamate-induced currents at a holding potential of -60 mV (Fig. 2a). Curve fits of the data by the Hill equation showed that the Hill coefficients strongly depended on extracellular glutamate concentration: the Hill coefficient was 1.2 at 1.0 mM glutamate and 2.0 at 0.2 mM glutamate. At lower glutamate concentrations, the curves apparently display even larger Hill coefficients.


Figure 2: Na to glutamate stoichiometry. a, Na dependence of glutamate-induced current. The data obtained for 0.2 and 1 mML-glutamate were fitted to the Hill equation. Hill coefficients increased with decreasing glutamate concentrations. b, Na uptake (20 min) in the presence and absence of L-glutamate. Oocytes expressing EAAC1 showed large glutamate-induced Na uptakes in the presence of either 1 or 0.2 mML-glutamate. A low level of Na uptake was also observed in the absence of glutamate demonstrating the Na leak phenomenon of EAAC1. c, comparison of Na uptake and [C]glutamate uptake in the same batch of EAAC1 cRNA-injected oocytes. The Na to glutamate flux ratios were 2.1 to 1 for 1 mM glutamate and 2.3 to 1 for 0.2 mM glutamate. This demonstrates that two Na ions are cotransported with each glutamate molecule. Na and [C]glutamate uptakes were measured in the presence of 40 mM Na, 1 mM ouabain, 0.1 mM amiloride, and 0.1 mM bumetanide.



Because the Hill coefficient depended on extracellular glutamate concentration (L-Glu), the Na to glutamate coupling was reinvestigated using a different approach. Briefly, the initial rates of the [C]glutamate and Na uptakes were directly compared. Ouabain, amiloride, and bumetanide were added to the bath to inhibit endogenous oocyte Na transport. Oocytes injected with EAAC1 cRNA exhibited large glutamate-induced Na uptakes (Fig. 2b). Water-injected control oocytes did not show significant Na uptake. EAAC1 mediated a low level of Na uptake in the absence of glutamate (Fig. 2b, right), which is consistent with a Na leak phenomenon, in analogy to that of the Na/glucose cotransporter(20) . The difference between the Na uptake obtained in the presence and absence of glutamate was taken as the glutamate-induced Na uptake (Fig. 2c). The initial rates of [C]glutamate uptake obtained L-Glu of 1 and 0.2 mM were determined on the same batch of EAAC1 cRNA-injected oocytes using the approach illustrated in Fig. 1a. The rates of the Na and [C]glutamate fluxes are compared in Fig. 2c. The ratios of the fluxes were 2.1:1 at 1.0 mML-Glu and 2.3:1 at 0.2 mML-Glu. The Na to glutamate coupling ratio is therefore 2:1 at both 1.0 and 0.2 mML-Glu. This indicates that the Na to glutamate coupling ratio does not depend on the extracellular glutamate concentration as suggested by Hill equation analysis.

EAAC1-mediated glutamate transport in oocytes was associated with intracellular pH (pH) changes as determined by monitoring the pH of oocytes using pH-sensitive microelectrodes. In the experiment shown in Fig. 3a, application of 100 µML-glutamate to EAAC1 cRNA-injected oocytes caused pH to decrease from 7.58 to 7.50 and the oocyte membrane to depolarize. A similar result was obtained using L-aspartate as a substrate, whereas leucine which is not a substrate of EAAC1 did not result in intracellular acidification. On average, the application of 100 µML-glutamate elicited a dpH/dt of -10.0 ± 1.1 10 pH units/s and a depolarization of 39.8 ± 2.4 mV (n = 25). These values allow the calculation of the OH/H to charge coupling (Fig. 3b). The changes in pH and the membrane depolarization were converted to currents by the following equations: I = [dpH/dt] F z, and I = V/R, where F is the Faraday constant, the buffering power (15 mM/pH unit), z = 1, and R the input resistance of an oocyte which was (1 M). Assuming an oocyte volume of 0.25 µl, the result is an estimated OH/H to charge coupling of 0.9 ± 0.1:1. These values indicate that transport of 1 L-glutamate molecule is associated with the cotransport of either 1 H ion or the countertransport of 1 OH ion.


Figure 3: H/OH coupling and K dependence. a, acidic amino acids transported by EAAC1 acidify and depolarize oocytes. The effect of 100 µM of either L-glutamate, L-leucine, or L-aspartate on intracellular pH (pH) and membrane potential (V) were measured using microelectrodes. 100 µM of either L-glutamate or L-aspartate rapidly depolarize and acidify oocytes while L-leucine has no effect. b, calculated charge to OH or H coupling. Plotted are the currents calculated from either the dpH/dt or the depolarization elicited by 100 µML-glutamate. The OH/H to charge coupling ratio is 0.9 ± 0.1. c, inhibition of L-glutamate-induced current by external K. Currents evoked by 50 µML-glutamate were measured at various K concentration (bath medium 0 mM-50 mM) at holding potentials of -30 and -60 mV and normalized to the current measured on the same oocyte (the value at 0 mM K is 100%). Each point represents the mean ± S.E. from six oocytes. The abscissa indicates external K concentration on a logarithmic scale.



Extracellular K inhibited glutamate-induced currents in voltage-clamped oocytes injected with EAAC1 cRNA, as previously reported(1) . In Fig. 3c, the inhibition is compared at -60 and -30 mV. It is greater at -30 mV than at -60 mV, consistent with K countertransport.

Reversed Glutamate Transport

Using the two-electrode voltage clamp method the reversed operation of EAAC1 was observed as an outward current. This was achieved in the presence of high extracellular K and by eliminating extracellular Na. In control experiments, glutamate-evoked inward currents were observed when using the high Na and low K solution in the perfusate (Fig. 4a). In the experiments addressing reversed glutamate transport, it was assumed that the intracellular glutamate concentration in oocytes is 10 mM, in analogy to neurons(21) , and the experiment was therefore started by counter-inhibition with 10 mML-Glu (Fig. 4b). Removal of glutamate resulted in a significant outward current, due to efflux of glutamate driven by the outwardly directed electrochemical gradients of Na and glutamate. In support of this interpretation, the outward current was enhanced by depolarization (Fig. 4b). No significant currents were observed in water-injected control oocytes. Fig. 4c shows the current-voltage relationship determined by stepping the voltage between -150 and +50 mV. The figure further confirms that the current is strongly enhanced by depolarization.


Figure 4: Reversed transport of EAAC1. a, ``normal'' glutamate transport. Glutamate evoked currents were determined in voltage-clamped oocytes injected with EAAC1 cRNA under normal ion-gradient conditions (2 K and 96 mM Na in the bath). b, reversed transport under disrupted ion-gradient conditions (98 mM K and no Na in the bath). Due to the coupling stoichiometry of EAAC1, the high extracellular potassium and the absence of extracellular Na causes EAAC1 to run in reverse. The experiment was initiated by trans-inhibition with bath applied 10 mM glutamate, assuming that the intracellular glutamate concentration is 10 mM. Removal of glutamate resulted in an outward current which is due to release of glutamate out of the cell driven by the intracellular Na concentration. The outward current was enhanced by depolarization, consistent with Na-driven reversed transport. c, current-voltage relationship of reversed glutamate transport based on voltage jump experiments. Oocytes expressing EAAC1 were clamped at -50 mV, and voltage steps were applied while bathed in medium containing either 96 mM K and 10 mM glutamate and 0 mM Na or 96 mM K without glutamate and NaCl.



Voltage Jump Studies

This approach was used to study the presteady-state and steady-state characteristics of EAAC1 in response to voltage jumps and to determine the kinetic parameters of EAAC1-mediated glutamate transport as a function of membrane potential.

Presteady State Currents

In contrast to the Na/glucose cotransporter SGLT1(9) , no significant presteady-state currents were observed in oocytes expressing rabbit EAAC1 in the presence of Na and in the absence of L-Glu (data not shown). A similar lack of presteady-state currents was observed for human EAAC1 (2).

Steady-state Currents

The I-V curves for the currents evoked by L-glutamate in the concentration range of 1 µM-1 mM demonstrated that the currents do not reverse between -150 to + 50 mV (Fig. 5). The currents saturated with hyperpolarization only at L-Glu below 40 µM whereas there was no saturation at higher L-Glu.


Figure 5: Current-voltage relationship of EAAC1-mediated glutamate uptake based on voltage jump studies. The oocyte membrane was held at a holding potential of -50 mV and the following six test potentials were applied: -150, -110, -70, -30, +10, and +50 mV. Currents due to the application of L-glutamate (1 µM to 1 mM) were recorded, and those currents obtained in the absence of glutamate were subtracted. The extracellular concentrations of K was 2 mM and of Na 98 mM.



Apparent K and I values obtained at different extracellular Na (Na) concentrations were plotted as a function of membrane potential (Fig. 6). As previously observed for human EAAC1, the K and I values determined at 100 mM Na both increase with membrane hyperpolarization (Fig. 6)(2) . In the present study we have determined the voltage dependence of K and I at reduced Na. Fig. 6a shows that re-ducing extracellular Na reverses the voltage dependence of K and causes it to increase to exceedingly high values with depolarization.


Figure 6: Dependence of K and I on extracellular Na concentration. The voltage dependence of K (a) and I (b) were determined at extracellular Na-concentrations of 10, 30, 50, and 100 mM using the voltage jump method.




DISCUSSION

The structure-function analysis of transport proteins requires knowledge of their transport mechanisms and the molecular rearrangements that occur during the transport process. Site-directed mutagenesis has been a highly valuable approach in elucidating the structure-function relationship of ion channels. The success of these studies was enhanced by the availability of functional models describing the transport process and the associated molecular events. These models were introduced by Hodgkin, Huxley, and Katz and were further refined by several investigators (see Ref. 22 for review). To embark on structure-function analysis of ion-coupled solute transporters, the availability of such models would be useful. To this end, we performed a detailed analysis of the coupling stoichiometry and transport kinetics of rabbit EAAC1, and we discuss a hypothetical kinetic model of EAAC1-mediated glutamate transport.

Stoichiometry

The studies presented in Fig. 1reveal a glutamate to charge flux ratio of 1: 1.19 and demonstrate that one net positive charge is translocated with each glutamate molecule. Although the stoichiometry of Na-coupled transporters is commonly determined based on the Hill equation (see Ref. 23), this approach did not yield reliable information on the coupling stoichiometry of EAAC1. Similar problems were encountered when using this approach for the determination of the stoichiometry of the Na/glucose cotransporter SGLT1(24) . Therefore, the Na to glutamate coupling ratio was determined by comparing the initial rates of Na and [C]glutamate uptake. The Na to glutamate coupling ratio was close to 2:1 at extracellular glutamate concentrations of either 1 or 0.2 mM.

EAAC1-mediated transport was furthermore associated with a decrease in intracellular pH (pH) (Fig. 3a). The OH or H to charge coupling ratio was estimated from the rate of pH decrease. This calculation depends on the assumed oocyte volume of 0.25 µl(19) , an oocyte membrane resistance of 1 M and a buffering power () of 15 mM/pH unit(18) . The computed coupling ratio of 0.9 ± 0.1 is consistent with a 1:1 ratio for the H/OH to charge stoichiometry.

Taken together, these data give an overall stoichiometry of 1 glutamate: 2 Na: 1 OH/H: 1 charge. Since we also demonstrated that transport mediated by EAAC1 depends on K and since Attwell and colleagues (8) proposed that high affinity glutamate transport is coupled to the counter transport of OH or other pH-changing ions such as HCO, and not to the cotransport of H, we infer that EAAC1-mediated glutamate transport is coupled to the cotransport of 2 Na ions and the countertransport of 1 OH (or HCO) ion and 1 K ion. This stoichiometry is unlike that proposed by Stoffel and colleagues (25) for the glial glutamate transporter GLAST. Based on Hill equation analysis, these investigators suggested that GLAST is coupled to the cotransport of three Na-ions. The overall stoichiometry of EAAC1, however, corresponds to that proposed by Attwell and co-workers (8) for high affinity glutamate transport in salamander retia glia cells.

Most previous studies addressing the stoichiometry of high affinity glutamate transporters were based on kinetic analysis(8, 25) . However, as discussed above (see Fig. 2), this technique can yield ambiguous results, in particular in cases where complex cooperativities between different substrate binding sites occur. Nevertheless, two reports were based on the analysis of the fluxes of glutamate ([C]glutamate), Na (Na), and K (Rb)(37, 38) , yielding partial information on the stoichiometry. The present work constitutes the first determination of the overall stoichiometry of a high affinity glutamate transporter which is based solely on comparison of the fluxes of the individual transporter substrates.

Based on this stoichiometry and the ionic concentration gradients which exist across plasma membranes, the concentrating capacity of EAAC1 in neurons and epithelial cells and the ratio of intra- and extracellular glutamate concentrations which can be attained at equilibrium can be estimated. Assuming that the intracellular glutamate concentration in neurons is 10 mM, the stoichiometry predicts an equilibrium extracellular glutamate concentration of 0.6 µM(8) . This value is well within the range of the prevalent glutamate concentration of the cerebrospinal fluid. Our results therefore verify that EAAC1 has the capacity to contribute to the maintenance of the low extracellular glutamate concentration in the central nervous system.

Reversed Glutamate Transport

In facilitated transporters such as the glucose transporter GLUT1 (26) and the urea transporter UT2 (19), it is generally accepted that net transport of substrates across cell membranes proceeds from the outside to the inside or from the inside to the outside, depending on the concentration gradients of the transport substrates across the cell membrane. By contrast, in ion-coupled transporters, net transport is considered to be unidirectional at given electrochemical ion gradients that energize uphill solute transport. However, if the ionic gradients are altered net transport of ion-coupled transporters can occur in the opposite direction. This was demonstrated by Attwell and co-workers (27, 28) for glutamate transport in salamander retina glia cells. In the present study, we show that EAAC1 expressed in Xenopus oocytes can run in the reversed direction. The I-V relationships of the outward currents obtained by two different procedures (Fig. 4, b and c) were essentially identical and demonstrated that the currents are highly voltage-dependent. Reversed glutamate transport currents become evident at potentials above -70 mV and steadily increased with depolarization. They did not saturate with increasing the membrane potential up to +50 mV (Fig. 4c). This I-V curve, when rotated by 180°, is similar to that of the ``forward'' glutamate transport (Fig. 5). This suggests that the reversed mode of EAAC1-mediated transport is a true reversal of the overall forward operation of transport and that the stoichiometry, the basic mechanism of transport and the rate-limiting step are likely to be the same.

The demonstration of EAAC1-mediated reversed glutamate transport suggests that the disrupted ion gradients which occur during pathologic conditions, such as ischemia after a stroke or hypoxia, could cause EAAC1 to run in reverse resulting in a non-vesicular release of glutamate into the synaptic cleft. The strong and widespread expression of EAAC1 in neurons throughout the CNS()suggests that EAAC1 could contribute to the rise in glutamate to neurotoxic levels during pathological conditions. Glial-reversed glutamate transport may also contribute to the extracellular rise, but the neuronal contribution is predicted to be larger because neurons have a significantly higher content of glutamate than astrocytes(29, 30) . Astrocytes convert glutamate to glutamine by glutamine synthetase, an enzyme which is lacking in neurons(31) . Thus, it is probable that reversed glutamate transport mediated by neuronal EAAC1 appears to be of particular pathologic consequence.

Charge Movement and Rate-limiting Steps of EAAC1-mediated Glutamate Transport

EAAC1 did not exhibit significant current relaxation in response to voltage jumps. A plausible interpretation of this behavior is that the empty carrier [C]` (see Fig. 7) is electroneutral and that any conformational changes of EAAC1 which occur during the transport process do not involve movement of charged amino acid residues within the membrane electric field. This would further suggest that the fully loaded carrier [CNaGlu]` (intermediate 4 in Fig. 7) has one positive charge and that its translocation is electrogenic and thereby voltage-dependent.


Figure 7: Hypothetical kinetic transport model of EAAC1. The model predicts that the kinetics of transport are ordered and that the transporter has a cation-binding site to which either Na or K binds and an anion-binding site to which either Glu or OH binds. Loading of the carrier at the extracellular surface is predicted to involve binding of the first Na, following glutamate binding which then allows binding of the second Na. The complex then translocates to the inside. This process is called the charge translocation step (conversion of intermediates 4-5) and is predicted to be rate limiting at an extracellular Na concentration of 100 mM. The relocation step (translocation of intermediate 10) is predicted to be accelerated by electroneutral countertransport to 1 OH and 1 K ion. Partial reactions such as the Na leak are not indicated in this diagram.



In analogy to human EAAC1(2) , rabbit EAAC1 exhibited large steady-state currents in the presence of extracellular L-glutamate (1 µM to 1 mM) in response to voltage steps (Fig. 5), and currents were inwardly directed and asymptotically approached zero near approximately +50 mV. Further in analogy to human EAAC1, the voltage dependence of the currents was affected by the extracellular glutamate concentration. At lower glutamate concentrations (below 30 µM), the current saturated as the membrane hyperpolarized and exhibited a sigmoidal dependence on membrane voltage. At higher glutamate concentrations, the current did not saturate, even if the membrane was hyperpolarized to -150 mV. This substrate-dependent current-voltage relationship is distinct from that of other transporters such as SGLT1 (32) which was sigmoidal for all substrate concentrations tested. This suggests that glutamate modulates the function of EAAC1, i.e. through a shift of the rate-limiting step.

At an extracellular Na concentration of 100 mM, the apparent Michaelis constant for L-glutamate (K) and the maximal transport rate I of rabbit EAAC1 both increased with hyperpolarization (Fig. 6). This result is congruent with findings of human EAAC1 (2) according to which both K and I increased with hyperpolarization and exhibited a linear relationship. We previously concluded based on this observation that K and I must be determined by a single, voltage-dependent step and that this step must correspond to the charge translocation step, i.e. the translocation of the fully loaded carrier [CNaGlu]`(2). The strong voltage dependence of the glutamate evoked current of rabbit EAAC1, the lack of saturation by hyperpolarization at glutamate concentrations >40 µM (Fig. 5), and the simultaneous increase in both K and I at 100 mM Na (Fig. 6) all support the view that the rate-limiting step of rabbit EAAC1 is also the voltage-dependent charge translocation step. Thus, in both rabbit and human EAAC1, the K values appear to be determined by the charge translocation step and not by glutamate binding.

Important new insights into mechanistic aspects of EAAC1 were obtained from studies addressing the dependence of glutamate transport on extracellular Na concentration (Na). Reduction of Na changed the voltage dependences of K and I entirely. At Na of 30 mM or less, K no longer decreased but increased with depolarization to exceedingly high values, whereas I became less voltage-dependent. This indicates that, at reduced Na, another step which is less voltage-dependent becomes rate-limiting, e.g. binding of Na or glutamate to the extracellular surface.

Cooperativity of Na and Glutamate Binding

The Hill equation gives information on the cooperativity of the substrate-binding sites. For Na-coupled solute transporters, the Hill coefficient often reflects the Na to substrate coupling ratio. However, as pointed out by Kimelberg et al.(33) the analysis is only valid if the affinities for the different Na-binding sites are similar. If there is a coupling ratio of 2:1 and the second Na binds with an affinity 10-100 times greater than that of the first Na, the function becomes essentially hyperbolic because it predominantly reflects the Na dependence of the low affinity Na-binding site. Fig. 2a demonstrates that the Hill coefficient increase from 1.2 to >2 with decreasing glutamate concentrations, whereas Na and [C]glutamate uptake studies reveal that the increase in Hill coefficients with decreasing L-Gluis not associated with a change in the Na to glutamate coupling ratio. The lack of correlation between the Hill coefficients and the true coupling ratio therefore indicates that EAAC1 has Na-binding sites with different affinities. The effect of L-Glu on the sigmoidicity of the Na dependence of transport furthermore suggests that glutamate increases the affinity for Na, i.e. the second Na (Fig. 7). Consistent with this, the effect on the sigmoidicity was particularly strong when L-Glu was near or below the apparent K of L-glutamate (Fig. 2a) which was around 30 µM (Fig. 6a). The Na leak phenomenon which was demonstrated by a low level of Na uptake in the absence of L-Glu (Fig. 2a) indicates that even in the absence of L-Glu, the second Na ion can bind with low affinity, resulting in translocation of the putative [CNa] complex (see Fig. 7).

Based on the Hill equation analysis of EAAC1, we hypothesize that binding of L-glutamate increases the affinity for Na binding (e.g. binding of the second Na ion). If L-Glu indeed modulates Na binding, this would be consistent with the finding that saturation of currents with hyperpolarization is only observed at low L-Glu (Fig. 5). At low L-Glu (<40 µM), it is likely that the rate-limiting step is related to substrate binding, i.e. binding of Na to the extracellular surface, whereas at high L-Glu, the charge translocation step is rate limiting and the current steadily increases with hyperpolarization.

Hypothetical Kinetic Model

A kinetic transport model of EAAC1 which is based on the present data, previous studies of human EAAC1 (2), and studies of brush border membrane vesicles (35) is presented in Fig. 7. The model includes ordered transport kinetics. EAAC1 is predicted to have a cation-binding site for either two Na ions or one K ion and an anion-binding site for either one glutamate ion or one OH ion. The cation-binding site is proposed to have two Na-binding sites with different affinities for Na. After binding of the first Na, glutamate binds, followed by binding of the second Na, in analogy to the model for the Na/glucose cotransporter proposed by Bennet and Kimmich (34; see also ref. 23). The fully loaded carrier (intermediate 4) translocates and the substrates are released to the cytoplasm. K and OH then bind to the cation- and anion-binding sites, respectively, and the transporter recycles to the outside, releasing K and OH to the extracellular medium. As suggested by Kinne and colleagues(35) , we hypothesize that the charge translocation step (translocation of intermediate 4) is rate limiting and that the relocation step (relocation of intermediate 10) is electroneutral. In ion-coupled solute transporters, the relocation step of the empty carrier is often considered to be rate-limiting (see Ref. 2). In EAAC1, countertransport of K and OH is predicted to serve to increase the rate of recycling of the transporter to such an extent that the charge translocation step becomes rate-limiting. In contrast, recycling of the empty carrier is thought to be rate limiting for the Na/glucose cotransporter(9, 32) . However, to absorb glutamate efficiently as an important metabolite in epithelial cells and to terminate glutamate's neurotransmitter action at glutamatergic synapses, glutamate transporters must have a high turnover rate. The required speed may be obtained by electroneutral coupling of the recycling step to energetically favorable K and OH countertransport.

Our data provide new insights into the molecular events that are associated with the transport process of EAAC1. A central question that remains to be addressed is which parts of EAAC1 are involved in glutamate translocation. The extended hydrophobic stretch of EAAC1 (residues 357-439) is of particular interest because it is highly conserved among all prokaryotic and eukaryotic members of the EAAC1 family(36) . This region is predicted to span the membrane at least three times (putative transmembrane domains 8-10). Due to its hydrophobicity, however, it is conceivable that it is largely embedded in the membrane and forms a flexible structure which constitutes the charge translocation pathway. An important role of putative membrane spanning domains 1-7 may be to anchor this translocation pathway in the membrane.


FOOTNOTES

*
This work was supported by the National Institute of Health Grants DK-43171 (to M. A. H.) and DK-30344 (to W. F. B.), the International Human Frontier Science Program Organization Long-Term Fellowship Award (to Y. K.), and the National Kidney Foundation Fellowship Award (to M. F. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Pharmacology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181, Japan.

To whom correspondence should be addressed: Renal Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-5850; Fax: 617-732-6392.

The abbreviations used are: Na, extracellular Na concentration; L-Glu, extracellular glutamate concentration.

Y. Kanai, P. G. Bhide, M. DiFiglia, and M. A. Hediger, manuscript submitted.


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