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Journal of Physiology (1991), 436, pp. 169-193 With 14 figures Printed in Great Britain

ELECTROGENIC UPTAKE OF GLUTAMATE AND ASPARTATE INTO GLIAL CELLS ISOLATED FROM THE SALAMANDER (AMBYSTOMA) RETINA

BY BORIS BARBOUR, HELEN BREW AND DAVID ATTWELL From the Department of Physiology, University College London, Gower Street, London WClE 6BT

(Received 20 April 1990) SUMMARY

1. The effects of excitatory amino acids on the membrane current of isolated retinal glial cells (Muller cells) were investigated using whole-cell patch clamping. 2. L-Glutamate evoked an inward current at membrane potentials between -140 and +50 mV. The current was larger at more negative potentials. 3. The glutamate-evoked current was activated by external cations with relative efficacies: Na+ > Li+> K+ > Cs+, choline. It was activated by internal cations with relative efficacies K+ > Rb+ > Cs+ > choline. Chloride and divalent cations did not affect the glutamate-evoked current. 4. Raising the intracellular sodium or glutamate concentrations, or raising the extracellular potassium concentration, reduced the current evoked by external glutamate. The suppressive effect of internal glutamate was larger when the internal sodium concentration was high. 5. Some analogues of glutamate also evoked an inward current. Responses to Laspartate resembled those to glutamate, but for aspartate the apparent affinity was higher and the voltage dependence of the current was steeper. In the physiological potential range the current evoked by a saturating dose of aspartate was less than that evoked by a saturating dose of glutamate. 6. The uptake blocker threo-3-hydroxy-DL-aspartate (30,tM) reduced the glutamate-evoked current, but also generated a current itself. Dihydrokainate (510 /LM) weakly inhibited the glutamate-evoked current without generating a current itself. 7. The commonly used blockers of glutamate-gated ion channels, 2-amino-5phosphonovalerate (APV; 100 ,M), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 /bM), and kynurenate (1 mM) had no effect on the glutamate-evoked current. 8. The voltage dependence, cation dependence and pharmacological profile of the current evoked by excitatory amino acids indicate that it is caused by activation of the high-affinity glutamate uptake carrier. This carrier appears to transport one glutamate anion into the cell, one K+ ion out of the cell, and two or more Na+ ions into the cell, on each carrier cycle. At the inner membrane surface some or all of the transported Na+ dissociates from the carrier after the transported glutamate has dissociated. MS 8441

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9. In addition to glutamate, the uptake carrier can also transport aspartate and threo-3-hydroxy-DL-aspartate, but not dihydrokainate. INTRODUCTION

L-Glutamate is a major neurotransmitter throughout the brain, and in the retina of lower vertebrates it may be the transmitter employed by photoreceptors and bipolar cells (Miller & Slaughter, 1986). The actions of glutamate on neurones are well known. Usually, glutamate opens cation channels, which have a rather poor selectivity between sodium and potassium and typically have a reversal potential around 0 mV (Hablitz & Langmoen, 1982; Mayer & Westbrook, 1984). In addition, glutamate has been shown to open chloride channels in invertebrate neurones and muscle cells and in vertebrate cones (Szczepaniak & Cottrell, 1973; Cull-Candy, 1976; Sarantis, Everett & Attwell, 1988). The actions of glutamate on glial cells have received much less attention. Glial cells play a crucial role in the operation of glutamatergic synapses. There are no extracellular enzymes to terminate glutamate's synaptic action (unlike for acetylcholine at the neuromuscular junction). Instead, uptake of glutamate into glial cells maintains a low extracellular concentration of glutamate, allowing removal of glutamate from the synaptic cleft by diffusion in the extracellular space. The properties and function of glutamate uptake are reviewed by Hertz (1979) and Erecinska (1987). The uptake of glutamate is driven by the co-transport of sodium ions down their electrochemical gradient. If, for example, two sodium ions are transported into the cell with each glutamate anion (Baetge, Bulloch & Stallcup, 1979; Stallcup, Bulloch & Baetge, 1979), one would expect uptake to be associated with a current flow into the cell. Such a current flow would allow glutamate uptake to be monitored electrically. The main type of retinal glial cell, the Muller cell, is known to take up glutamate strongly from the extracellular space (White & Neal, 1976; Ehinger, 1977). We have reported recently that, in Muller cells from the salamander retina, glutamate evokes an inward current, apparently associated with glutamate uptake, and that this inward current depends on external sodium and internal potassium (Brew & Attwell, 1987; Barbour, Brew & Attwell, 1988). In this paper we describe in detail the properties of this current. Use of the whole-cell patch-clamp technique to record from Muller cells has allowed us to investigate the dependence of the uptake current on the ionic composition of the solution inside and outside the cell, and on membrane potential. The ability to study glutamate uptake while voltage-clamping the cell offers a significant advance over other techniques for studying uptake since, as will be shown in this paper, the membrane potential is a key determinant of the rate of uptake. METHODS

Muller cells were isolated from the retina of the salamander (Ambystoma tigrinum or Ambystoma mexicanum; no difference was observed between results from the two species), using a modification of the methods described by Bader, MacLeish & Schwartz (1979). Half a retina was incubated for 30 min in 2 ml of a solution containing (mM): NaCl, 66; glucose, 15; NaH2PO4, 10; KCl, 3-7; NaHCO3, 25; sodium pyruvate, 1; cysteine, 10; and papain, 4 units/ml. The half-retina was then

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washed by dropping it through 2 ml of ordinary Ringer solution 4 times, and was then drawn in and out of a Pasteur pipette (the tip size of which had been reduced by heating in a Bunsen burner flame) until the retina broke up into individual cells. The cells were plated onto the glass bottom of the recording chamber. Muller cells were easily recognized by their distinctive size and shape (Fig. 1). Cells were recorded from within 4 h of isolation. The normal Ringer solution used to superfuse the cells contained (mM): NaCl, 105; KCl, 2-5; CaCl2, 3; MgCl2, 05; glucose, 15; HEPES, 5; pH adjusted to 7-3 with 2 mM-NaOH. Barium Ringer solution had 6 mM-BaCl2 added to this, to block the cells' potassium conductance (Newman, 1985). Other external solutions were based on modifications of these solutions, as described in the text. The bath electrode used for electrical recording was normally a silver chloride pellet, but was a 4 M NaCl agar bridge for experiments where the external chloride concentration was changed. Patch pipettes used for whole-cell clamping typically had a resistance of 1 MQ2 (when immersed in the superfusion solution), leading to a typical series resistance (measured from the current response to a voltage jump) of 2 MQ when in whole-cell mode. Voltage errors due to the series resistance were usually less than 1 mV. Pipettes were usually filled with a 'standard internal medium' consisting of (mM): KCl, 80; potassium acetate, 15; NaCl, 5; HEPES, 5; MgCl2, 7; Na2ATP, 5; CaCl2, 1; K2EGTA, 5; pH adjusted to 7 0 with 14 mM-KOH; or were filled with a 'chloride internal medium' for which potassium acetate in the above recipe was replaced by KCl. Results obtained with these two solutions were similar. Other internal solutions were based on modifications of these recipes, as described in the text. Junction potentials were corrected for as described by Fenwick, Marty & Neher (1982). Normally, L-glutamate or L-aspartate (or a related compound) was added to the external solution. Occasionally, glutamate was ionophoresed from pipettes containing 1 M-sodium glutamate (adjusted to pH 8-0 with NaOH). The concentration of free glutamate or aspartate in the superfusion solution is reduced from its nominal value by binding to divalent cations. In the presence of Mg2+, Ca2+ and Ba2+ the fraction of amino acid unbound is given approximately by: [free amino acid]/[total amino acid] = 1/(1 +KMg[Mg2 ]total+Kc8[Ca2[]C0a +KB,[Ba ]tota) where KMg, KCa and KB. are the stability constants for divalent cations binding to the amino acid. This expression is correct to within 03% for [glutamate] or [aspartate] < 100 /M. For 1 mM [glutamate] or [aspartate], this expression is in error by < 1 and < 2-7 % respectively, but these errors are irrelevant at such high amino acid concentrations where the uptake carrier is completely saturated with amino acid. For glutamate and aspartate this fraction has the values 89 and 82 %, respectively, in the absence of barium, and 81 and 75% in the presence of barium (stability constants taken from Martell & Smith, 1974). Thus, for a nominal glutamate concentration of 30 /IM (the value used in most of our experiments), the actual free glutamate concentration is 24 or 27 CM in the presence or absence of barium: adding barium reduces the concentration by 10 %. For simplicity in the text we quote the nominal concentrations of glutamate and aspartate. The input resistance of isolated Muller cells in normal Ringer solution is about 10 MCQ (Mobbs, Brew & Attwell, 1988). Consequently, to polarize the cells significantly from their resting potential of around -90 mV, large currents must be passed, leading to large series resistance errors, and to the membrane current becoming noisy because of large currents flowing through the cell's potassium channels which fluctuate open and closed (Brew, Gray, Mobbs & Attwell, 1986). Thus, to investigate the voltage dependence of the glutamate-evoked current over a wide voltage range, it is desirable to reduce the cell conductance. We did this by blocking the cell's potassium channels with 6 mM-barium (Newman, 1985). This increases the cell's resistance to about 200 MQ, and greatly increases the voltage range over which the cell can be polarized. Figure 2 shows the current response of an isolated Miller cell voltage clamped at -80 mV to the superfusion of 30 ,lMglutamate, initially in normal Ringer solution. Glutamate evokes an inward current of about 280 pA which, we will argue below, is due to the activation of glutamate uptake. Figure 2 shows that barium evokes an inward current shift at -80 mV, due to the potassium channels being blocked, but only slightly reduces the magnitude of the glutamate-evoked current (mean (± S.E.M.) reduction was 4-7 + 1-1 % in five cells). A small reduction in glutamate-evoked current is expected because 6 mM-barium binds roughly 10 % of the nominally 30 /M-glutamate present (see above): at a nominal glutamate concentration of 30 1UM, on the dose-response curve shown in Fig. 4B this would lead to a 4 3 % reduction of the current. Below (Fig. 3) we show also that barium has no effect on the voltage dependence of the glutamate-evoked current.

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Apical membrane

Cell

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Fig.

1. A

Miiller

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showing the cell body (where the usually attached), and the vitreal endfoot (which contains over 90% of the cell membrane's potassium conductance). Sometimes the process of cell isolation led to cells losing their endfoot, allowing recording of the glutamate-evoked current over a wide potential range without needing to block the cell's potassium conductance with barium (see Fig. 3).

whole-cell electrode

was

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The magnitude of the glutamate-evoked current varied considerably from one Miller cell to another. This complicated the interpretation of experiments described below in which we wished to compare the responses of different cells voltage clamped with patch pipettes containing different solutions. A plot (data not shown) of the current evoked at -40 mV in forty-four cells by 30 uMglutamate as a function of cell capacitance indicated that much of the variation in glutamateevoked current reflects variation in cell size. The current varied over a 5-fold range, but was positively correlated with cell capacitance and thus with surface area (correlation coefficient of the regression line, r = 0 803). The fractional scatter (standard deviation/mean value) in the values of glutamate-evoked current per unit area of cell membrane (measured as the ratio of the current to the capacitance) was about half the fractional scatter in the values of the current. Using this result, we assessed the relative magnitude of different cells' responses to glutamate by comparing values of evoked current divided by capacitance for each cell (rather than by simply comparing the currents). RESULTS

Glutamate and voltage dependence of the glutamate-evoked current Dependence of the current on membrane potential As shown in Fig. 2, glutamate evokes an inward membrane current in Muller cells. If this current resulted from glutamate opening ion channels, one would expect this current to become less inward with depolarization, and to be outward above the channel reversal potential (typically 0 mV for glutamate-gated cation channels). If, on the other hand, the glutamate-evoked current reflects glutamate uptake into the cell (associated with an inward charge movement due to an excess of Na+ ions cotransported with each glutamate), an inward current is predicted at all potentials: although the net current generated by the uptake carrier must in principle show a reversal potential, the change of current detected when glutamate is applied to the outside of the cell will always be inward. In normal external solution the low resistance of the Muller cell (10 MQ) precludes investigation of the glutamate-evoked current over a large voltage range. To overcome this problem, two different methods were used. First, 6 mM-barium was added to the external solution to block the cells' potassium conductance (see Methods). In Fig. 3A the response to ionophoresed glutamate is shown for a Muller cell in barium Ringer solution. Glutamate evokes an inward current at all the potentials shown. The current is smaller at more positive potentials, but is still inward near the Nernst potential for sodium (+ 49 mV). At potentials above + 50 mV, no current change could be detected in response to glutamate. The second method employed to investigate the glutamate-evoked current over a wide voltage range was to use Muller cells which had lost their vitreal endfeet (Fig. 1) in the cell isolation procedure. The endfoot contributes only a small fraction of the glutamate-evoked current, because it is a small fraction of the cell area and because the glutamate response per unit area of membrane is small in the endfoot (Brew & Attwell, 1987). However, the endfoot membrane contains over 90% of the resting potassium conductance (Newman, 1984; Brew et al. 1986), so cell bodies without the endfoot have resistances above 100 MS in normal Ringer solution (no barium). Figure 3B shows that the voltage dependence of the glutamate-evoked current in such a cell body (in barium-free solution) is similar to that in the whole cell of Fig. 3A studied in barium. The current data of Fig. 3A and B, normalized by cell capacitance, are plotted as

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a function of voltage in Fig. 3D where they approximately superimpose. These data establish that the glutamate-evoked current is still inward at a potential 50 mV more positive than the reversal potential of most glutamate-gated channels (around 0 mV). We considered it possible that the inward current might still reflect the

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Fig. 2. Glutamate evokes an inward membrane current, which is little affected by the 6 mM-barium used in many experiments to block the cells' potassium channels. Cell whole-cell clamped to -80 mV. Glutamate (Glu, 30 /SM) is initially applied in barium-free Ringer solution. Then barium is applied, producing an inward current shift due to a block of outward potassium current. In the presence of barium, glutamate evokes an inward current similar in magnitude to that seen in the absence of barium. Later, when the barium has been washed out, glutamate again produces an inward current in normal Ringer solution.

opening of ion channels by glutamate if, unusually, the channels were largely permeable to calcium. To test this, we recorded from a Muller cell body in a solution similar to normal (non-barium) Ringer solution but with the normal 3 mM-calcium chloride replaced by 1 mM-CaCl2 and 5 mM-Na2EGTA which buffers the external calcium concentration to a calculated 2-2 x 10- M, i.e. lower than the calcium concentration in the patch pipette (10' M) so the Nernst potential for calcium was -19 mV. The voltage dependence of the glutamate-evoked current in this solution (Fig. 3 C and D) was the same as that in normal or barium Ringer solution. A clear inward current was generated at +29 mV for the cell of Fig. 3 C, well above the calcium reversal potential. Thus, the voltage dependence of the glutamate-evoked current is consistent with the inward current being produced by electrogenic glutamate uptake (and with uptake being reduced at positive potentials). The

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Fig. 3. Voltage dependence of the glutamate-evoked current. A, current responses of a whole Muller cell, clamped to the voltages shown by each record, to ionophoresed glutamate (timing shown by upper trace). Ringer solution contained 6 mM-barium to block the cell's potassium conductance. B, responses of a Muller cell body which had lost its endfoot (containing most of the cell's potassium conductance) during the cell isolation procedure. No barium was present in the Ringer solution. C, responses of a Muller cell without an endfoot in Ringer solution containing no barium and essentially no calcium (see text). D, peak currents from A, B and C plotted as a function of voltage. For each cell the currents have been normalized by cell capacitance, to compensate approximately for differences in cell size. The resulting I-V relations roughly superimpose.

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voltage dependence of the current is not consistent with it being produced by glutamate opening ion channels.

Dependence of the current on external glutamate In Fig. 4A we plot the magnitude of the current evoked by different concentrations of superfused glutamate at five different membrane potentials in the same cell mv

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Fig. 4. Glutamate dependence of the glutamate-evoked current. A, magnitude of the inward current (ordinate) evoked in one cell by different concentrations of superfused glutamate (abscissa) at different membrane potentials (shown by each curve). Experiment performed in barium Ringer solution. Curves fitted by eye. B, dose-response data in A normalized to the currents produced at each potential by 1 mM-glutamate. Curve is a Michaelis-Menten relation with a Km of 19-8 /M. C, best-fit values of Km obtained from Lineweaver-Burke plots of the data in A. Line has the mean value of 19-8 /M.

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(inward current is plotted upwards for convenience on this graph). At all voltages the current rises monotonically to reach a maximum value at 1 mM-glutamate. Experiments on other cells showed that no further increase in current was obtained on changing from 1 to 2 mM-glutamate. The data in Fig. 4A are replotted in Fig. 4B after normalization to the current produced by 1 mM-glutamate. The dose-response curves for the different voltages are essentially superimposable, implying that the voltage dependence of uptake is the same at all glutamate concentrations. Lineweaver-Burke plots for the data at each voltage were used to obtain best-fit values of apparent Km for a Michaelis-Menten curve

[glutamate]0

[glutamate]0 +Km' The resulting values of Km are shown in Fig. 4C. There is no systematic dependence of Km on voltage between -100 and -20 mV. The mean value of Km over this range is 19-8 /tM (or 16 /M allowing for divalent cations reducing the free glutamate concentration: see Methods). A Michaelis-Menten curve with this Km is drawn through the data in Fig. 4B. This relatively low value of Km implies that the glutamate uptake mechanism being studied is high-affinity glutamate uptake.

'Inactivation' of the glutamate-evoked current Sometimes, when glutamate was applied for up to 30 s, the inward current generated was sustained, with little change of current magnitude occurring while glutamate was present. Often, however, the current evoked by large doses of glutamate (e.g. 1 mM) showed a pronounced sag. With prolonged application the current magnitude at the end of the response could be only half that when glutamate was first applied. Figure 5 shows an example of this. After a brief application of 30 /LM-glutamate, 1 mM-glutamate was applied for 3 min. The resulting current decreased from its initial peak value, quickly at first and then more slowly. Subsequent applications of 30 /SM-glutamate generated responses smaller than that elicited before 1 mM-glutamate was applied, which gradually recovered in size towards the initial value. In addition, an outward current shift (unusually large for the cell of Fig. 5) occurred after the long application of 1 mM-glutamate; this also slowly decayed away. We tentatively suggest that this inactivation of the glutamate-evoked current is a result of the accumulation of sodium or glutamate inside the cell. As is shown below, raised intracellular sodium or glutamate reduce the glutamate-evoked current (because sodium or glutamate can rebind to the carrier, keeping it longer at the intracellular face of the membrane in this situation). The outward shift in current baseline occurring after the 1 mM-glutamate application in Fig. 5 could result from activation of the electrogenic sodium-potassium exchange pump by a raised [Na+]i, or from the glutamate uptake carrier running backwards when sodium and glutamate have accumulated inside the cell.

Dependence on internal glutamate If the glutamate-evoked current is produced by glutamate uptake, we would expect a high concentration of glutamate inside the cell to reduce the current: in this

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B. BARBOUR, H. BREW AND D. ATTWELL

situation it will be more difficult for the uptake carrier to lose glutamate at the inner membrane surface, so the return of the carrier to the outer membrane surface (to pick up more glutamate) will be slowed. On the other hand, if glutamate evokes a current by opening channels, there is no reason to expect internal glutamate to affect the current size.

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1 min Fig. 5. 'Inactivation' of the glutamate-evoked current. At -40 mV, a test pulse of 30 /LMglutamate was applied, evoking an inward membrane current. Then, prolonged application of 1 mM-glutamate evoked a larger current, which became smaller with time. After removal of the 1 mM-glutamate, the baseline current in the absence of glutamate was more outward, and the response to 30 /tM-glutamate was depressed for several minutes.

Figure 8 shows that when the sodium concentration in the patch pipette is above 20 mm, replacing 55 mm-KCl in the patch pipette by 55 mM-potassium glutamate decreases by 70 % the current evoked by 30 /M-external glutamate at -40 mV, consistent with a reduction of glutamate uptake as predicted above. In addition, we will show below that the degree of suppression of the current by internal glutamate depends critically on the sodium concentration in the cell, as can be predicted from the fact that both sodium and glutamate are transported by the uptake carrier. Ion dependence of the glutamate-evoked current Dependence on external sodium and related ions We have already reported that replacement of external sodium by choline suppresses the glutamate-evoked current (Brew & Attwell, 1987). In Fig. 6A-C we show experiments in which the external sodium was completely replaced by various monovalent cations: lithium, potassium and caesium. The relative amplitudes (mean + S.E.M.) of the currents evoked at -40 mV by 30 /tM-glutamate in the presence of these ions were INa ILi IK ICs Icholine = 1:0-011+ 0-002 (n = 4): 0-008 + 0-008 (n = 4): 0 (n = 3): 0 (n = 7). Thus, as has been found from radiotracing studies of

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glutamate uptake (Kanner & Sharon, 1978), uptake is highly selective for external sodium. The dependence of the current on external sodium concentration (sodium replaced by choline) is plotted in Fig. 6D. The current depends on [Na']. in a sigmoid manner, suggesting that more than one Na+ ion is transported by the carrier. The A

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[Na+lo (mM) Fig. 6. Dependence of glutamate uptake on external monovalent cations. A, membrane current of a Miuller cell at -40 mV during repeated application of 30 /LM-glutamate in the superfusate (timing shown by filled bars at bottom), and while sodium ions in the external solution were completely replaced by lithium (top bar). B, similar experiment to that in A, but with external Na+ replaced by K+. Raising the external potassium concentration generates a large inward current, presumably through the small fraction of potassium channels that remain unblocked by 6 mM-barium (although the slow onset of this current is not understood). C, similar experiment to that in A, but with Na' replaced by Cs'. Lithium, potassium and caesium support very little uptake current. D, the dependence of uptake current (produced by 30 /,M-glutamate at -40 mV) on external sodium concentration (sodium replaced by choline: mean+S.E.M.. data from 3, 5, 11 and 5 cells in 10, 20, 30 and 50 mM [Na+]O normalized to response in 107 mm [Na'].). Smooth curve through the points is the cube of a Michaelis-Menten relation, i.e. Vmax {[Na']/ ([Na+]0+K)}3. with Vmax = 1-77 and K = 22-5 mm. Dashed curve is a Hill plot with the form ax{[Na+]}2/({[Na+}2 +K2) where Vmax = 1-218 and K = 50 mm.

data could be fitted roughly by a power of a Michaelis-Menten relation, or by the Hill equation (see Fig. 6D), but the glutamate-evoked currents recorded below 10 mM [Na+]O were too small to determine accurately whether the current is proportional to the 2nd, 3rd or a higher power of [Na+]O.

B. BARBOUR, H. BREW AND D. ATTWELL

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Dependence on internal sodium Since the glutamate uptake carrier transports sodium into the cell (Baetge et al. 1979; Stallcup et al. 1979), one would expect that raising the sodium concentration inside the cell (by filling the patch pipette used for whole-cell recording with a high A

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Fig. 7. Inhibition of glutamate uptake by internal (patch pipette) sodium. A, increasing the interal sodium concentration (replacing choline) decreases the glutamate-evoked current in cells clamped to - 40 mV. Data shown are mean+ S.D. for five cells at each value of [Nailpipette relative to five cells studied with 0 mm~v [Na+]PiPette,,. B, specimen I-V relations for the current induced by 30 fSm-glutamate in one cell studied with 0 mm [Nal]pipette and in another with 100 mm~[Na+]pipette. Data are normalized by the capacitance of each cell (423 and 360 pF for the 0 and 100 mm~v [Nliet cells respectively).

sodium concentration solution) would decrease the current evoked by glutamate. Figure 7A shows that this is indeed the case. For these experiments a modified internal medium was used, consisting of (mM): NaCl, x; choline chloride, 100-x; HEPES, 5; CaCl2, 1; MgCl2, 7; MgATP, 5; K2EGTA, 5; KOH, 15; pH = 7; where [Na+]i was x = 0, 15, 40, 60 or 100. For each value of sodium concentration in the patch pipette, the response to 30 /tM-glutamate at -40 mV was measured in five cells. After normalizing by cell capacitance (see above), the mean current thus obtained was then divided by the mean current obtained in five cells studied with 0 mM [Na+]pjpette on the same day. When the sodium concentration in the pipette was large, the glutamate response was smaller, with a reduction to one-half occurring at approximately [Na+]pipette = 60 mM. Raising internal sodium also alters the voltage dependence of the glutamateevoked current (Fig. 7B). High [Na+]i produces a much greater fractional suppression

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of uptake at depolarized potentials than at hyperpolarized potentials. A similar change in the shape of the I-V relation is observed when uptake is reduced by lowering the external sodium concentration (Brew & Attwell, 1987).

Interaction of internal sodium and glutamate The internal sodium concentration was found to have a dramatic effect on the suppression of glutamate-evoked current by internal glutamate which was described 2.5 mM

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Fig. 8. Inhibition of the glutamate-evoked current by intracellular glutamate depends on the internal sodium concentration. A, specimen records from four different Miiller cells showing the effect on the current evoked at - 40 mV by 30 Sm-glutamate (ifilled bars) of raising the concentration of glutamate in the patch pipette (and hence presumably in the cell) from 0 (left pair of records) to 55 mm (right pair of records), for a patch pipette sodium concentration of 2-5 mm (top records) and 40 mm (bottom records). Currents have been normalized by cell capacitance to compensate for differences in cell size. B, dependence on pipette sodium concentration (abscissa) of the suppressive effect of internal glutamate. The ordinate is the ratio (mean+ S.D.) of the current evoked by 30 /Mglutamate when the pipette contained 55 mM-glutamate to that evoked with no glutamate in the pipette, i.e. comparing the size of the left- and right-hand records in experiments like those in A. Pipettes contained (mM): KCl or potassium glutamate, 55 (to give [glutamate] = 0 or 55 mM); NaCl, x (where x was 0, 2-5, 5, 7 5, 10, 20 or 40); choline chloride, 40-x; HEPES, 5; CaCl2 1; MgCl2, 7; MgATP, 5; K2EGTA, 5; KOH, 12-5; pH = 7. above. Figure 8A (bottom) compares the glutamate responses of two cells studied with 40 mm [Na+]pipette, one with no glutamate in the pipette and the other with 55 mM-glutamate in the pipette. Internal glutamate greatly decreases the response as described above. However, at the top of Fig. 8A the same experiment is carried out on cells using only 2-5 mm [Na+] in the patch pipette. In this situation, internal

B. BARBOUR, H. BREW AND D. ATTWELL

182 A

B 500

20 ,M-glutamate

(6)

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400

(6) 300

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-40 mV 30,uM-glutamate

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5 mM

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120 220 260 80 Time after patch rupture (s)

0

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20

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80

60

100

[K+]pipette (mM)

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.(10).

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Rb+ t J

IL

-600

95

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100 pA

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Fig. 9. The dependence of the glutamate uptake current on internal monovalent cations. A, when recording from a cell with a pipette containing no potassium, the current evoked at -40 mV by externally applied glutamate (20 uM) gets progressively smaller with time after rupturing the patch membrane (to go to whole-cell recording mode). Extrapolation of the glutamate-evoked current backwards in time suggests that much of the current decay occurs in the first 40 after patch rupture. B, dependence of glutamate-evoked current (normalized by cell capacitance to compensate for variations in cell size) on pipette potassium concentration (potassium replaced by choline). Data shown are mean + s.E.M. for the number of cells shown by each point. Smooth line is a Michaelis-Menten relation with an apparent Km = 15 mm (fitted from a Lineweaver-Burke plot). C, specimen I-V relations for the current evoked by 30 #M-glutamate in one cell with 95 mm [K+]pipette and another with 5 mm [K4]pipette. Data normalized by cell capacitance (315 and 243 pF for the cells with 95 and 5 mm [K4]pipette respectively) to compensate for differences in cell size. D, specimen records showing that with all the internal (patch pipette) potassium replaced by rubidium (top) or caesium (bottom), 30,uM-glutamate (filled bars) still produces a substantial uptake current at -40 mV. s

ELECTROGENIC GLUTAMATE UPTAKE

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glutamate has almost no suppressive effect. Figure 8B shows the response with 55 mM-glutamate in the pipette relative to that with no glutamate in the pipette, as a function of [Na+]pipette. The suppressive effect of internal glutamate increases in a sigmoid manner with [Na+]pipette, so that below 5 mm [Na+]pipette there is almost no suppression, but at higher [Na+]pipette internal glutamate does reduce the response, reaching a maximum suppression of about 70% (i.e. I(55 mm [glutamate]pipette)/ I(0 mm [glutamate]pipette) = 0 3) when [Na+]pipette = 40 mM. This interaction of the suppressive effects of internal sodium and glutamate can be explained as a result of both sodium and glutamate being transported by the uptake carrier (see Discussion).

Dependence on internal potassium and related ions Kanner & Sharon (1978) have suggested, on the basis of radiotracing experiments, that glutamate uptake is activated by internal potassium ions. To investigate this, we recorded from Muller cells using a patch pipette solution in which the potassium was replaced by choline. The solution contained (mM): KCl, x (x = 0-100); choline chloride, 100- x; HEPES, 5; MgCl2, 7; MgATP, 5; CaCl2, 1; Na2EGTA, 5; pH adjusted to 7 with 14 mM-NaOH. To avoid the possibility of potassium flowing in across the cell membrane and accumulating inside the cell when it is held at a negative holding potential (and thus making [K+]i higher than [K+]pipette), the external Ringer solution used had the normal 2-5 mM-KCl omitted to abolish any potassium influx through the small fraction of channels left unblocked by barium. (Control experiments (see Barbour et al. 1988) showed that removal of external potassium had no effect on the glutamate-evoked current when [K+]pipette was high.) In this situation, when [K+]pipette was zero the glutamate-evoked current decreased to almost zero in the first few minutes following transition to whole-cell recording (Fig. 9A). This decline did not occur when [K+]pipette was high, suggesting that intracellular potassium activates glutamate uptake in these cells. However, although the glutamate-evoked current was small with no potassium in the patch pipette, it was still significantly greater than zero, possibly because the intracellular [K+] had not been reduced to zero, or because some uptake is possible in the absence of potassium. In the steady state (i.e. after about 3 min when exchange of the cell and pipette solutions had occurred) the current evoked by 30 ,uM-glutamate at -40 mV showed a roughly Michaelis-Menten dependence on [K+]pipette with a Km of 15 mm (Fig. 9B), Lowering [K+]pipette had only a small effect on the voltage dependence of the glutamate-evoked current (Fig. 9C), the current being reduced slightly more by low [K+]pipette at positive than at negative voltages. Activation of the glutamate-evoked current by other internal cations was studied using the same external solution (0 [K+]0, 6 mM-barium) and internal solution as described above but with internal KCl replaced by CsCl or RbCl. Caesium and rubidium were found to be less effective activators of the current (Fig. 9D). At -40 mV, 30 /SM-glutamate produced currents (normalized by cell capacitance) with the different cations of relative magnitudes (mean+ S.D.) IK :IRb:ICs :Ichoine= 1 :0 86+0-14 (n = 4):0-66+0-17 (n = 4):0 04+0 03 (n = 10).

B. BARBOUR, H. BREW AND D. ATTWELL

184 A

B

Gluconate Chloride Chloride

mV -100

-50

r

0

50

I1

External anion o Gluconate * Chloride

/ o

_ -100

0'

200 pA7

pA

/-200 A

,0 _ _ 30 uM-glutamate

/

-300

mV

-100

-50

Pipette anion o Gluconate * Chloride

0

50

e -200

e o

- -400

-1000

M -

-1200

Fig. 10. Effect on the glutamate-evoked current of changing the extra- and intracellular chloride concentration (cells studied in barium Ringer solution). A, membrane current of a Muller cell at -43 mV for which the external chloride was replaced by gluconate (top bar), while glutamate was repeatedly applied (bottom filled bars). Chloride removal had no effect on the magnitude of the glutamate-evoked current at -43 mV. B, magnitude of current evoked by 30 /LM glutamate in another cell at different voltages, with either chloride or gluconate as the main external anion. Over the voltage range studied, chloride removal had no effect on the shape of the I-V relation. C, currents produced by 30 zMglutamate at various voltages in two different Muller cells with chloride or gluconate as the main pipette anion. Currents from the two cells were normalized by cell capacitance (265 pF for the cell studied with internal chloride and 230 pF for the cell studied with gluconate) to compensate for differences in cell size.

Dependence on external calcium and magnesium The possible role of divalent cations in glutamate uptake was assessed from response of cells to ionophoresed glutamate, in external Ringer solution (no barium) containing various calcium concentrations. Calcium levels of 1, 3 and 10 mm were obtained by including either 1, 3 or 10 mm-CaCl2 in the recipe for normal Ringer

ELECTROGENVIC GLUJTAMATE UPTAKE

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solution given in the Methods section. To obtain '0 mm' calcium, 3 mM-CaCl2 in that recipe was replaced by 1 mm-CaCl2 plus 5 mm-Na2EGTA, leading to calcium being buffered at 22 x 10-8 M. There was no significant variation in response size (at -80 mV) with [Ca21]. over the range investigated (n = 3, data not shown: see also Fig. 3C and D). Similarly, removing magnesium from the external solution had no effect on the magnitude of the current (three cells studied in Ringer solution containing barium; data not shown).

Dependence on anions The main anion, both intracellularly and extracellularly, in most of our experiments, was chloride. Any chloride dependence of the glutamate-evoked current is of interest, because a chloride-dependent glutamate uptake system has previously been reported (Pin, Bockaert & Recasens, 1984; Waniewski & Martin, 1984). Figure 10A and B show an experiment in which the dependence of the glutamate-evoked current on external chloride was tested. All the chloride in barium Ringer solution was replaced by the much larger gluconate anion. This had no effect (seven cells) on the magnitude of the glutamate-evoked current at -43 mY (Fig. IOA), nor on the voltage dependence of the current (Fig. lOB). To test the dependence of the glutamate-evoked current on intracellular chloride, cells were recorded from sequentially, alternating between six cells with chloride internal medium (see Methods) in the pipette and seven cells with a pipette medium in which all the chloride was replaced by gluconate. On average at -40 mV 30 1aMglutamate evoked a current (per unit capacitance) of 0 59 + 0-02 pA/pF (mean + S.E.M.) with chloride inside and 0-52 + 0-05 pA/pF with gluconate inside, i.e. not significantly different. Replacing internal chloride by gluconate had no effect on the voltage dependence of the glutamate-evoked current (Fig. 10C).

Effects of aspartate and other glutamate analogues We have reported previously (Brew & Attwell, 1987) that the response of Muller cells to glutamate analogues shows a pharmacological profile appropriate for glutamate uptake. Thus, L-glutamate and L- and D-aspartate evoke a large current, while kainate, NMDA, quisqualate and D-glutamate evoke a negligible current. In this section we investigate in more detail the effects of L-aspartate, a putative central nervous system transmitter, the effects of threo-3-hydroxy-DL-aspartate and dihydrokainate, known blockers of glutamate uptake, and the effects of three blockers of glutamate-gated channels, 2-amino-5-phosphonovalerate (APV), 6cyano-7-nitroquinoxaline-2,3-dione (CNQX) and kynurenate. In preliminary experiments using barium Ringer solution, the glutamate uptake current was little affected by dihydrokainate, APV and CNQX. To make sure that this was not because of barium binding to these glutamate analogues, reducing their effective concentration (as occurs to a small extent for glutamate and aspartate, see Methods), we then applied these agents (and also kynurenate, which was not tested in barium Ringer solution) in barium-free Ringer solution. Removal of barium did not unmask any large effects of the analogues.

B. BARBOUR, H. BREW AND D. ATTWELL

186

The effect of L-aspartate Qualitatively, L-aspartate generates an inward current similar to that produced by glutamate. Quantitatively, however, there are three important differences. First, the dose-response curve for aspartate has a maximum agonist-evoked current that is 8 1

A

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. 6-

Aspartate

0.6-

PMM 20 pm---

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0-4-

0)

r

50

pA

1000

l 1 min

MM--- {-0-l Glutamate 0

10

20

30

100

[Aspartate] (pM)

Fig. 11. Comparison of the dose-response curves for currents evoked by L-glutamate and L-aspartate in a Muller cell in barium Ringer solution. A, membrane current of a cell at -40 mV during perfusion of 6, 20 and 1000 uM-aspartate and glutamate. B, dose-response curves for the currents evoked by different doses of aspartate in another cell voltage clamped to -65 ([1), -40 (@) or -15 mV (0). Inward currents evoked by I00 4Maspartate at these potentials were 132, 66 and 28 pA respectively. Smooth curve through the points is a Michaelis-Menten relation with a Km of 3-8 ,M (fitted from a Lineweaver-Burke plot).

different from that for glutamate. In Fig. llA, the current evoked at -40 mV by a saturating dose of aspartate is less than half that evoked by a saturating dose of glutamate. Second, the apparent affinity for aspartate is higher than for glutamate: in Fig. II A, 6 /tM-glutamate evokes less than half the maximum glutamate-evoked current, while 6 /IM-aspartate evokes more than half the maximum aspartate-evoked current. In Fig. 11 B, the shape of the dose-response curve for aspartate is approximately independent of voltage, as for glutamate, but the apparent Km of 3-8 /tM is much lower than we generally find for glutamate (Km = 8-20 /M). Third, the voltage dependence of the current evoked by aspartate is steeper than that for glutamate. Figure 12A shows that, for 30 /M of each drug, the uptake current is similar at - 120 mV, and that glutamate evokes a larger current than aspartate at more positive potentials but a smaller current than aspartate at more negative potentials. Averaged data for the ratio of the currents evoked by 30/M glutamate and aspartate, as a function of voltage, are shown in Fig. 12B. Applying 30 /SM-glutamate and 30 /tM-aspartate simultaneously evoked an inward

ELECTROGENIC GLUTAMATE UPTAKE

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current that was larger than that produced by 30 ftM-aspartate alone, but smaller than that produced by 30 4uM-glutamate alone (Fig. 12C). The implications of this result are considered in the Discussion.

The effect of threo-3-hydroxy-DL-aspartate (THDA) This glutamate analogue has been shown to reduce glutamate uptake (Balcar & Johnston, 1972). In Fig. 13A we show that at -43 mV this agent not only reduces A

B

mV

-150

-100

-50

0

uA

1.5 r

-100

1.00

pA 0.5 [

-200

0L1 -150

-300

-50

-100

0

mV C

mV

Asp

+

Glu

Glu

-150

Fig. 12. A, voltage dependence of the currents evoked by 30 ,uM-aspartate and 30/Mglutamate in a Muller cell (in barium Ringer solution). B, ratios of the aspartate- and glutamate-evoked currents at different potentials in experiments like that in A (data are mean + S.E.M. from four cells, except for the potentials -130, 120 and 0 mV for which data are from two, three and two cells respectively). C, currents evoked in a Muller cell (in barium Ringer solution) by 30 /SM-aspartate, 30,uM-glutamate and 30 ,uM-aspartate plus 30 /M-glutamate. -

to 30 /SM-glutamate, but also generates an inward current itself. The THDA-evoked current (30 #M, -43 mV) was 29+6% (S.E.M., n = 6) of the current evoked by 30 lM-glutamate in the absence of THDA, and THDA reduced the glutamate-evoked current to 26 ± 5 % (mean + S.E.M., n = 6) of its control value.

the

response

mean

188

B. BARBOUR, H. BREW AND D. ATTWELL A

100

THDA

pA[

L-GIu

L-GIu

L-GIu

100 s

DHK

B

L-GIu

L-GIu

100 pA

L-GIu

[

I

30 s Fig. 13. The effect of glutamate uptake blockers on the glutamate uptake current. A, at -43 mV 30 4M-threo-3-hydroxy-DL-aspartate (THDA) induces an inward current and inhibits the current evoked by 30 ,#M-glutamate. These experiments were carried out in barium Ringer solution. B, 510 ,uM-dihydrokainate (DHK) reduces the magnitude of the current evoked by 30 /tM-glutamate at -80 mV. This experiment was carried out in Ringer solution without barium (see text).

These results suggest that threo-3-hydroxy-DL-aspartate is itself transported by the glutamate uptake carrier, and that it blocks glutamate uptake by competing for sites on the uptake carrier. The effect of dihydrokainate In rat brain slices this glutamate analogue has been shown to inhibit glutamate uptake with an IC50 of 176 /tM (Johnston, Kennedy & Twitchin, 1979). In three cells, dihydrokainate (510 #M) was found to reduce by 15 + 1 % (mean + S.E.M.) the current

ELECTROGENIC GLUTAMATE lUPTAKE 189 produced at -83 mV in a Muller cell by 30 ,SM-glutamate, without generating any inward current itself (Fig. 13B). This suggests that dihydrokainate has a lower affinity for the salamander uptake carrier than for that in rat, and that it blocks uptake by binding without being transported itself. The effects of competitive glutamate antagonists CNQX, APV and kynurenate are widely used as blockers of glutamate-gated channels. None of these compounds had a significant effect on the glutamate uptake current. In 20 ,uM-CNQX the mean current evoked by 30 JLM-glutamate at -83 mV was 95+2% (S.E.M., n = 2) of its control value. For 100 /SM-APV the value was 102 + 1 % (n = 3) and for 1 mM-kynurenate, 98 + 2 % (n = 3). DISCUSSION

Effects of glutamate on glial cells: glutamate uptake and ion channels The work of Bowman & Kimelberg (1984) and Kettenmann & Schachner (1985) suggested that glutamate could depolarize glial cells by activating an electrogenic carrier. Subsequent experiments have demonstrated that glutamate generates an inward current by opening ion channels in some cultured astrocytes (Sontheimer, Kettenmann, Backus & Schachner, 1988; Usowicz, Gallo & Cull-Candy, 1989) and in freshly isolated glial progenitor cells (Barres, Koroshetz, Swartz, Chun & Corey, 1990). The pharmacology, voltage dependence and inhibition by intracellular glutamate of the glutamate-evoked current we observe in salamander Muller cells strongly suggest that it is produced by the activation of glutamate uptake, and not by ion channels. Activation of the current by intracellular potassium is also difficult to account for on the basis of glutamate opening ion channels. Different subclasses of glial cells may possess glutamate uptake carriers or glutamate-gated ion channels, according to function. In the cerebellum, type 2 astrocytes express glutamate-gated channels (Usowicz et al. 1989), while type 1 astrocytes show a glutamate-evoked current with a voltage dependence similar to that shown in Fig. 3 (Cull-Candy, Howe & Ogden, 1988) presumably reflecting glutamate uptake. The presence of glutamate uptake in glial cells is thought to maintain the extracellular concentration of glutamate below neurotoxic levels, and to help terminate the synaptic action of glutamate. The functional significance of glutamate-gated ion channels in glial cells is unknown.

Co-transport of Na+ ions on the glutamate uptake carrier The sigmoid form of the [Na+]. dependence of the current in Fig. 6D is consistent with the co-transport of more than one sodium ion with each glutamate anion. Most radiotracing work in glia has failed to observe a similar sigmoid dependence of glutamate uptake on [Na']0. For example, Drejer, Larsson & Schousboe (1982) show a sigmoid Na+ dependence for neurons (granule cell culture) but a Michaelis-Menten Na+ dependence for cultured astrocytes. However, direct measurement of sodium and glutamate fluxes found a sodium: glutamate transport ratio of 2 in neuronal and glial cultures (Baetge et al. 1979; Stallcup et al. 1979) or of 2-3 in cultured astrocytes (Kimelberg, Pang & Treble, 1989). Investigation of the equilibrium transmembrane

190

B. BARBOUR, H. BREW AND D. ATTWELL

distribution of D-aspartate, [Asp]i/[Asp]0, has shown that this varies approximately as the square of the sodium concentration gradient, {[Na+]0/[Na+]i}2, in synaptosomes (Erecinska et al. 1983) and in C6 astrocytoma cells (Erecinska, Troeger, Wilson & Silver, 1986), also suggesting a sodium :glutamate transport ratio of 2.

Counter-transport of K+ ions on the uptake carrier Glutamate radiotracing experiments suggested that the glutamate uptake carrier counter-transports potassium (Kanner & Sharon, 1978; Burckhardt, Kinne, Stange & Murer, 1980). However, in those experiments membrane potential was not controlled, making it difficult to be certain whether effects of [K+] changes were direct effects or caused by a membrane potential change. Erecinska et al. (1983, 1986) have interpreted the effects of [K+] changes on the distribution of aspartate across synaptosome and astrocytoma cell membranes as being solely due to changes in membrane potential: in fact, however, their data are entirely consistent with one K+ ion being carried out of the cell (and one net positive charge moving in) on each carrier cycle. Our voltage-clamp experiments show unequivocally that internal potassium and external potassium (Barbour et al. 1988) affect uptake directly. The simplest interpretation of our results is that one K+ ion is transported out of the cell on each carrier cycle (because of the first-order dependence on [K+]pipette in Fig. 9B), but direct proof of this K+ flux is still lacking.

Stoichiometry of the uptake carrier The inward current associated with glutamate uptake reflects the movement of positive charge into the cell on each carrier cycle. If only two Na+ ions moved in with each glutamate anion, and one K+ ion moved out, on each carrier cycle no current would be generated. Movement of three Na+ ions into the cell on the carrier (which would not be inconsistent with the dependence on [Na+]. in Fig. 6D), or of two Na+ ions and one H+ ion (as proposed by Erecinska et al. 1983, 1986) would account for the electrogenic nature of the uptake. Evidence from work on the apparently similar kidney glutamate transporter strongly supports the idea of proton co-transport with glutamate (Nelson, Dean, Aronson & Rudnick, 1983). Inhibition of the uptake current by intracellular Na+ and glutamate We tentatively attributed the sag seen in the glutamate-evoked current during prolonged application of glutamate to accumulation of sodium or glutamate inside the cell. A simple estimate, ignoring diffusive contact with the patch pipette, shows that this is a plausible explanation. For a glutamate-evoked current of I = 200 pA, a Muller cell volume of V = 8 x 10-15 m3 (i.e. a cylinder 100 ,m long and 10 #tm diameter), and a stoichiometry such that three Na+ ions are transported per elementary charge moved, the rate of change of intracellular sodium concentration is given by d [Na+]i/dt = 31/(VF) where F is the Faraday constant. From this equation, d [Na+]i/dt = 0-8 mM/s, while glutamate would accumulate at one-third of this rate (ignoring removal by metabolic utilization). Thus, after a minute the intracellular [Na+] would have risen by 48 mm, say from 15 to 63 mM, and according to Fig. 7A this would reduce the uptake current by about 40%.

ELECTROGENIC GLUTAMATE UPTAKE

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Raising the pipette glutamate concentration greatly reduced uptake when [Na+]i was high, but not when [Na+]i was low. A simple explanation for this is that, as shown in Fig. 14, at the intracellular face of the membrane some Na+ dissociates from the carrier after the transported glutamate has dissociated. In this situation, when Glu

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