©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Arachidonic Acid Inhibits a Purified and Reconstituted Glutamate Transporter Directly from the Water Phase and Not via the Phospholipid Membrane (*)

Davide Trotti (1), Andrea Volterra (1), Knut P. Lehre , Daniela Rossi (1), Ola Gjesdal , Giorgio Racagni (1), Niels C. Danbolt(§)

From the (1) Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, P. O. Box 1105 Blindern, N-0317 Oslo, Norway Centre of Neuropharmacology, Institute of Pharmacological Sciences, University of Milan, via Balzaretti 9, 20133 Milan, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glutamate is believed to be the major excitatory transmitter in the mammalian central nervous system. Keeping the extracellular concentration of glutamate low, the glutamate transporters are required for normal brain function. Arachidonic acid (AA) inhibits glutamate uptake in relatively intact preparations (cells, tissue slices, and synaptosomes (Rhoads, D. E., Ockner, R. K., Peterson, N. A., and Raghupathy, E. (1983) Biochemistry 22, 1965-1970 and Volterra, A., Trotti, D., Cassutti, P., Tromba, C., Salvaggio, A., Melcangi, R. C., and Racagni, G. (1992b) J. Neurochem. 59, 600-606). The present study demonstrates that the effect of AA occurs also in a reconstituted system, consisting of a purified glutamate transporter protein incorporated into artificial cell membranes (liposomes). The characteristics of the AA effect in this system and in intact cells are similar with regard to specificity, sensitivity, time course, changes in V, and affinity. AA-ethyl ester is inactive, suggesting that the free carboxylic group is required for inhibitory activity. When incubated with proteoliposomes, AA (300 µM, 15 min) mostly partitions to the lipid phase (lipid/water about 95:5). However, uptake inhibition is abolished by rapid dilution (6.5-fold) of the incubation medium (water phase), a procedure that does not modify the amount of AA associated with lipids. On the contrary, inhibition remains sustained if the same dilution volume contains as little as 5 µM AA, a concentration inactive before saturation of liposome lipids with 300 µM AA. The same degree of inhibition (60%) is obtained by 5 µM AA following preincubation with the inactive AA-ethyl ester (300 µM) instead of AA. The lipids apparently inactivate AA by extracting it from the water phase. The results suggest that AA acts on the transporter from the water phase rather than via the membrane. This could be true for other proteins as well since -aminobutyric acid uptake is similarly affected by AA.


INTRODUCTION

Glutamate is probably the major mediator of excitatory signals in the mammalian central nervous system (Fonnum, 1984). Not only is glutamate required for most aspects of normal brain function, but it is believed to have pathological implications as well, because glutamatergic overstimulation can damage neurons (Olney, 1990). Consequently, the extracellular concentration of glutamate has to be kept low (in the order of 1 µM). As the brain contains huge amounts of glutamate (about 10 mmol/kg, wet weight), powerful uptake systems are present in both glial cells and neurons (for review, see Danbolt (1994)). The driving forces of the uptake process are the membrane potential and the gradients of Na, K, and OH(Kanner and Sharon, 1978; Bouvier et al., 1992). A change in the function of these transporters may have significant effects on synaptic transmission (Mennerick and Zorumsky, 1994; Barbour et al., 1994). Despite their obvious importance, little is known about their regulation (Danbolt, 1994). Impaired uptake may play a role in epilepsy (During and Spencer, 1993) and in neurodegenerative diseases (Rothstein et al., 1993).

Arachidonic acid (a fatty acid consisting of a 20-atom-long carbon chain with 4 double bonds and a carboxylic group at one end, 20:4) has been reported to inhibit several sodium-coupled amino acid transporters including the uptake systems for glutamate, glycine, and GABA() (Rhoads et al., 1983; Chan et al., 1983; Yu et al., 1986; Barbour et al., 1989; Zafra et al., 1990; Volterra et al., 1992b; Lynch et al., 1994; Volterra et al., 1994). Arachidonic acid is released from both neurons (Dumuis et al., 1988, 1990, 1994; Lazarewicz et al., 1990; Sanfeliu et al., 1990) and glial cells (Stella et al., 1994) upon activation of glutamate receptors. Free fatty acids, including arachidonic acid, are known to accumulate in the brain under pathological conditions such as ischemia and seizures (Siesjö et al., 1989)

All of the previous studies on arachidonic acid inhibition of amino acid uptake have been based on intact cells or complex preparations such as tissue slices and synaptosomes. It could therefore be argued that the effect of arachidonic acid is not a direct action on the transporter itself or its lipidic environment but an indirect effect through other mechanisms. Arachidonic acid influences several molecules (such as the Na/K-ATPase (Okun et al., 1992; Caspers et al., 1993) and protein kinase C (Hardy et al., 1994)) that are known to affect glutamate uptake. Furthermore, arachidonic acid in high concentrations has detergent-like physicochemical properties and might damage cell membranes and subsequently impair the ion gradients driving the uptake process. The successful purification of a glutamate transporter (Danbolt et al., 1990) has made it possible to study the function of this protein in the absence of other proteins. The present report, which is based on highly or partly purified glutamate transporters reconstituted into artificial membranes (liposomes), demonstrates that the effect of arachidonic acid is similar to that reported in intact preparations and therefore does not require the presence of other proteins. We provide evidence that the glutamate transporter protein itself is sensitive to low micromolar concentrations of aqueous arachidonic acid.


EXPERIMENTAL PROCEDURES

Materials

L-[H]Glutamic acid (50 Ci/mmol) and [H]GABA were purchased from Amersham Corp. [5,6,8,11,12,14,15-H]arachidonic acid (210 Ci/mmol) was from DuPont NEN (Boston, MA). Sephadex G-50 fine was from Pharmacia Biotech Inc. Affi-Gel ( N-hydroxysuccinimide esters of cross-linked agarose) and hydroxylapatite were from Bio-Rad. Diethylaminoethyl (DEAE)-cellulose (DE52) was from Whatman (Maidstone, Kent, United Kingdom). CHAPS, fatty acids, wheat germ agglutinin as free lectin were from Sigma. Stock solutions of the fatty acids were made in dimethyl sulfoxide (MeSO) and aliquoted and stored at 20 °C. Wheat germ agglutinin was immobilized to agarose as described previously (Danbolt et al., 1992). All other reagents were of analytical grade or better.

Rapid Gel Filtration on Spin Columns

Spin columns have been used in this study both for reconstitution of purified glutamate and GABA transporters and for replacing the external medium of the liposomes with another medium. They have also been used to remove substances that interfere with protein measurement. Sephadex G-50 fine is swollen in buffer overnight and packed in plastic syringes (1 ml) from which the pistons have been removed and the outlets closed by cotton fiber. The columns are then centrifuged (1200 rpm, 240 g; 2 min; Heraeus-Christ Minifuge-T) to remove the void volume. After applying the sample (0.2 ml) and allowing it to sink into the gel, the columns are centrifuged again (2 min, 1740 rpm, 500 g). The conditions are adjusted using phenol red and blue dextran 2000 as low and high molecular mass markers, respectively. The efficiency of the spin columns is illustrated by the addition of [H]glutamate to the sample. The columns reduce the concentration about 5000-fold.

Purification and Reconstitution of Glutamate and GABA Transporters into Liposomes

This has been done as described previously (Danbolt et al., 1990, 1992). Briefly, crude rat brain plasma membranes were solubilized with a zwitterionic detergent (CHAPS) and centrifuged. The supernatant (CHAPS extract) was passed through a wheat germ agglutinin-agarose column, and the glycoproteins were eluted with N-acetylglucosamine. The glycoproteins (``partially purified transporter'') in this fraction were either reconstituted directly or further purified by chromatography on hydroxylapatite and DEAE-cellulose before reconstitution (``highly purified''). The first purification step results in about a 10-fold purification of both glutamate and GABA transporters with respect to protein, but removes virtually all low molecular mass species like ATP and endogenous lipids. After the second and third purification steps, one predominant band is seen on SDS-polyacrylamide gel electrophoresis, and the specific glutamate transport activity is increased 30-fold in spite of inactivation (Danbolt et al., 1990). This preparation contains the glutamate transporter GLT-1 (Pines et al., 1992; Levy et al., 1993), but neither the glutamate transporter GLAST (Lehre et al., 1995) nor any GABA transporters (Danbolt et al., 1990). For reconstitution, the fractions (containing 20 mM CHAPS and 0.1-0.2 mg of protein/ml) were mixed with 1.5 volumes of a phospholipid/cholate/salt mixture (Danbolt et al., 1990), incubated on ice, and gel filtered to remove detergent and sodium ions on spin columns (see above) equilibrated with a medium containing potassium phosphate. The liposomes form spontaneously during this gel filtration, and the buffer these columns are equilibrated with becomes the internal medium of the liposomes. The liposome suspension contained 40-80 µg of protein and 30 mg of phospholipids/ml.

In some experiments, liposomes were formed in the presence of labeled glutamate. 150 µl of phospholipid/cholate/salt mixture was mixed with 100 µl of buffer (no protein) and gel filtered on spin columns equilibrated with sodium phosphate buffer containing labeled glutamate. By collecting these liposomes on Millipore filters (0.45-µm pores) and comparing the radioactivity retained with that in the buffer, the internal volume of the liposomes was found to be in the order of 20 µl/ml of liposome suspension. The integrity of the liposome membranes after exposure to arachidonic acid was tested by incubating the liposomes with arachidonic acid (50, 125, or 300 µM) for up to 120 min and checking if arachidonic acid induced a glutamate leak from the liposomes ().

Determination of Glutamate and GABA Transport Activity

This was done essentially as described (Danbolt et al., 1990). Briefly, 20 µl of proteoliposomes, containing and suspended in 0.12 M potassium phosphate buffer, pH 7.4, were diluted into 500 µl of PBSG (140 mM NaCl, 10 mM sodium phosphate buffer, 1% (v/v) glycerol, pH 7.4). MeSO with or without fatty acids was included in the incubation medium. The final MeSO concentration was kept constant at 1% (v/v). The mixture was preincubated (unless stated otherwise, 15 min at room temperature) before starting the uptake reaction by adding 1.4 µCi of labeled amino acid and 3 µM valinomycin (a selective potassium ionophore to ensure a negative membrane potential). The liposomes were incubated (room temperature for 70 s with glutamate or 7 min with GABA, unless stated otherwise). The reaction was terminated by dilution in 2 ml of ice-cold PBSG and filtration through Millipore HAWP filters (0.45-µm pores). The filters were rinsed 3 times and dissolved in Filter Count (Packard) for liquid scintillation counting. Addition of nigericin (a nonselective potassium-sodium ionophore abolishing ion gradients) to the incubation medium resulted in a complete loss of transport activity (equivalent to omitting the liposomes from the mixture) and served as negative control.

When the liposomes were incubated with low concentrations of glutamate (below 100 nM), the uptake was fairly linear with time for 1 min, but when high concentrations (above 3 µM) were used, the transport was linear with time for much shorter periods. In this study, an incubation time of 2 s has been used for the determinations of kinetic parameters. This is the shortest time consistent with reproducible results using available equipment.

Dilution of Liposomes Preincubated with Arachidonic Acid

The liposomes obtained after reconstitution were loaded and suspended in the same potassium-containing buffer. By filtering the liposome suspension through another set of spin columns (as described above) equilibrated with PBSG, the free external potassium was removed from the proteoliposomes. The washed liposomes (still with potassium on the inside) were diluted 1 + 3 with PBSG containing arachidonic acid or arachidonic acid ethyl ester (0 or 300 µM, final concentrations) and incubated (15 min at room temperature). Aliquots of 80 µl of diluted preincubated liposomes were added to different volumes of uptake medium (0, 80, 160, 240, or 440 µl of PBSG with 1.4 µCi of labeled amino acid, valinomycin, and 1% MeSO) with arachidonic acid (0, 5, 15, or 300 µM). The reaction was terminated by dilution and filtration as above.

The amount of arachidonic acid dissolved in the liposome membranes was assessed by treating liposomes as above, but using tritiated arachidonic acid diluted with unlabeled arachidonic acid to 300 µM final concentration. The liposomes were diluted as above and centrifuged (Beckman TL-100 microultracentrifuge, 100,000 rpm for 15 min), and the radioactivity in pellet and supernatant were compared.

Protein Determination

Protein content was determined by the method of Lowry et al. (1951). Interfering substances (EDTA, CHAPS, and N-acetyl-D-glucosamine), when present, were removed from the samples by gel filtration on Sephadex equilibrated with 0.4% SDS as described previously (Danbolt et al., 1990). Bovine serum albumin was used as standard.


RESULTS

Inhibition of Glutamate Uptake by Arachidonic Acid

The effect of arachidonic acid was studied in preparations of glutamate transporters purified and reconstituted as described previously (Danbolt et al., 1990). The inhibition of glutamate uptake by arachidonic acid in these preparations is dose-dependent (Fig. 1) with more than 65% inhibition at 300 µM (15 min preincubation). The effect of 10 µM arachidonic acid was not significant. No differences were found between the partially and highly purified glutamate transporter preparations (Fig. 1). At 125 µM arachidonic acid, 14.2 ± 1.8, 23.8 ± 3.5, 45.7 ± 6.6, 57.6 ± 4.4, and 56.6 ± 5.2% inhibition was obtained after 1, 5, 10, 15, and 30 min, respectively. In the absence of arachidonic acid, the Vand Kwas 10.6 ± 0.9 nmol/min/mg of protein and 8.3 ± 0.45 µM, respectively (Fig. 2). Arachidonic acid (125 µM) reduced the Vby 50 ± 5% ( p < 0.01) and increased the affinity slightly, by 19 ± 4% ( p < 0.05).


Figure 1: Concentration-dependent inhibition of partially and highly purified glutamate transporters by arachidonic acid. Glutamate uptake activity was determined as described under ``Experimental Procedures'' with a 15-min preincubation time. The results are expressed as percent inhibition + S.E. of control uptake. Bars represent the average of three separate determinations done in triplicate. The inhibition observed was significantly different from control ( p < 0.01) for all arachidonic acid concentrations tested except 10 µM. No differences were noted between the partially and highly purified preparations.




Figure 2: Effect of arachidonic acid on the kinetics of glutamate uptake. Liposomes inlaid with partly purified glutamate transporters were preincubated (15 min) and incubated (2 s) with different concentrations of glutamate in the absence () and in the presence () of 125 µM arachidonic acid (see ``Experimental Procedures'' for details). The results are expressed as mean ± S.E. of four experiments performed in triplicate. The data were analyzed with the direct linear plot method (Cornish-Bowden, 1974).



Structure-Activity Study of Transporter Inhibition by Fatty Acids

The effect of arachidonic acid was compared with that of other long chain saturated and unsaturated fatty acids as well as arachidonic acid esters (Fig. 3). Neither the trans-unsaturated fatty acid elaidic acid (18:1) nor the saturated fatty acids stearic acid (16:0) and arachidic acid (20:0) inhibited glutamate uptake. However, all of the cis-unsaturated fatty acids tested (including oleic acid, which is a cis-unsaturated analogue of elaidic acid) did inhibit glutamate uptake. The order of potency was as follows: eicosapentaenoic acid (20:5) > arachidonic acid (20:4) > docosahexaenoic acid (22:6) > linolenic acid (18:3) > oleic acid (18:1). The differences in potency between the various cis-configured fatty acids seem to correlate with the degree of unsaturation present in the molecule and thereby with the degree of folding. ( cis-unsaturated fatty acids are bent, while trans-configured acids are linear.)


Figure 3: Inhibition of glutamate uptake by different fatty acids and arachidonate esters expressed as percent of the inhibition by arachidonic acid. All agents (100 µM) were incubated (15 min) with proteoliposomes. Then, glutamate was added and the mixture incubated for 70 s. Arachidonic acid inhibited glutamate transport by 32 ± 4.5% at 100 µM (100% in graph). SA, stearic acid; ARA, arachidic acid; EA, elaidic acid; OA, oleic acid; LA, linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid, AA-ME, methyl arachidonate; AA-EE, ethyl arachidonate; AA-CE, cholesteryl arachidonate. Results are mean + S.E. of three experiments performed in triplicate.



Upon modification of the carboxyl-group, arachidonic acid lost its inhibitory activity. Thus, arachidonic acid-methyl ester showed very low activity, while arachidonic acid-ethyl ester and cholesteryl-arachidonate were inactive.

Reversal of Inhibition of Glutamate Transporter Activity

Liposomes were loaded with potassium phosphate, suspended in PBSG, and preincubated (15 min) with 300 µM arachidonic acid. Then, glutamate uptake was determined by mixing the liposome suspension with 0, 1, 2, 3, or 5.5 volumes of [H]glutamate-containing uptake medium. 70 s later, the uptake reaction was stopped. Inhibition by about 60% was observed when the uptake medium contained arachidonic acid (). However, when the liposomes were diluted into uptake medium without arachidonic acid, the inhibitory effect of arachidonic acid was lost proportionally to the volume of the uptake medium. Surprisingly, dilution with only 5.5 volumes was sufficient to restore the transport activity. This 1 + 5.5 dilution would reduce the average arachidonic acid concentration to 46 µM, which is expected to give almost 20% inhibition (see Fig. 1). These experiments show the following three things. 1) The arachidonic acid effect is reversible. 2) The phospholipid membranes have not been damaged by a detergent-like effect of arachidonic acid, because, if they had, there would not have been any ion gradients to drive the uptake. The integrity of the liposomes is also demonstrated in . 3) The degree of inhibition cannot be predicted from the average arachidonic acid concentration (see below). This finding suggested that the partition of arachidonic acid between the lipid and aqueous phases might be important.

The Relative Importance of Arachidonic Acid in the Water and Lipid Phases

In order to determine the amount of arachidonic acid bound to the lipids after the above dilution of preincubated liposomes (), liposomes were preincubated (15 min) with tritiated arachidonic acid (mixed with unlabeled arachidonic acid to a total arachidonic acid concentration of 300 µM), diluted as above, and centrifuged (15 min) to separate the liposomes from the water phase. Surprisingly, 95% of the labeled arachidonic acid remained in the lipids after dilution and centrifugation. Because dilution removed transport inhibition but had little effect on the amount of arachidonic acid incorporated into the liposome membranes, the lipid-bound arachidonic acid must be less important than the arachidonic acid in the water phase.

Although the lipidic arachidonic acid that had accumulated in the membranes during the preincubation, did not affect the transport activity, it dramatically increased the sensitivity of the transport to aqueous arachidonic acid. Thus, as shown in Fig. 4, liposomes preincubated with 300 µM arachidonic acid (15 min) and diluted as above were strongly inhibited by as little as 5 µM arachidonic acid in the dilution medium. 5 µM arachidonic acid had no effect on nonpretreated liposomes (compare with Fig. 1). Interestingly, a similar increase in sensitivity to arachidonic acid was observed after preincubation with arachidonic acid-ethyl ester (Fig. 4), which in itself is completely inactive on glutamate transport (Figs. 3-5). Moreover, when arachidonic acid and its ethyl ester were added together (100 µM of each) in a competition experiment using liposomes that had not been pretreated, the inactive ester increased the effect of arachidonic acid rather than reducing it (Fig. 5).


Figure 4: Preexposure increases arachidonic acid potency. Liposomes loaded with potassium phosphate and inlaid with purified glutamate transporters were ( 1step) suspended in potassium-free PBSG (see ``Experimental Procedures'' for details) and incubated (15 min) with 300 µM arachidonic acid ( AA300), 300 µM arachidonic acid ethyl ester ( AA- EE300), or 0 µM arachidonic acid ( CTL). Then ( 2 step), the liposome mixtures were diluted (1 + 5.5) with PBSG containing labeled glutamate and either arachidonic acid (0 µM, CTL; 300 µM, AA300; 15 µM, AA15; 5 µM, AA5) or arachidonate ethyl ester ( AA- EE300) and incubated for 70 s. The results are expressed as percent of control ( CTL- CTL); mean + S.E. of four experiments performed in triplicate. One-way analysis of variance analysis shows that (*) AA300-AA300 is not significantly different from AA300- AA15, AA300- AA5, or AAEE300- AA5, which are all significantly different from CTL ( p < 0.01).




Figure 5: The inhibitory action of arachidonic acid on glutamate uptake is potentiated by arachidonic acid ethyl ester. Liposomes were preincubated (15 min) with either 100 µM arachidonic acid ( AA), 100 µM arachidonic acid ethyl ester ( AAEE), or the two in combination. Glutamate uptake activity was assessed as described under ``Experimental Procedures.'' The overall inhibition by AA + AAEE was reversed by >90% following dilution of the liposome suspension, demonstrating that the potentiation was not due to disruption of the liposome membranes (data not shown). The results represent mean + S.E. of three separate experiments each performed in quadruplicate.



Inhibition of GABA Uptake by Arachidonic Acid

The sensitivity of the partly purified and reconstituted GABA transporter was comparable with that of glutamate uptake. At 25, 50, and 100 µM arachidonic acid (15-min preincubation, uptake reaction terminated after 7 min), 10 ± 5, 26 ± 5, and 55 ± 7% inhibition was observed. Like glutamate uptake, the GABA uptake was not sensitive to 100 µM arachidonic acid ethyl ester, and the arachidonic acid induced-inhibition was completely reversed upon dilution (1 + 5.5), suggesting that the mechanism of inhibition may be the same for the two types of transporters.


DISCUSSION

The finding that arachidonic acid inhibits the uptake of glutamate at concentrations compatible with intact membranes, demonstrates that the inhibitory effect is a direct action on the transporter protein itself or its lipidic environment. The effect of arachidonic acid on the purified and reconstituted transporter shows characteristics very similar to those observed in cultured astrocytes and Müller cells. The time course of the inhibition closely resembles that previously reported in astrocytes (see Fig. 3 A in Volterra et al. (1992b)) with a characteristic fast onset (0-1 min), a slower rise (1-15 min in liposomes versus 1-10 min in astrocytes), and a plateau phase (after 15 min), indicating saturation of the effect with time. The relative potencies of different fatty acids and the changes in Vand Kalso fit with previous findings (Volterra et al., 1992a, 1992b; Barbour et al., 1989). Thus, the simple reconstituted system with a single protein residing in an artificial membrane, retains all the characteristics of arachidonic acid sensitivity. Therefore it is possible to exclude indirect actions of arachidonic acid such as changes in the ion gradients (inhibition of Na/K-ATPase, modification of ion channels, etc.), protein kinase C activation, formation of downstream mediators (enzymatic derivatives of arachidonic acid: eicosanoids), or changes in membrane lipid composition due to active esterification of arachidonic acid in phospholipids by acetyltransferase enzymes.

The prevailing view, as expressed in the cited literature (Rhoads et al., 1983; Yu et al., 1986; Barbour et al., 1989; Zafra et al., 1990; Volterra et al., 1992a, 1992b), is that arachidonic acid probably affects the lipidic environment of the transporters. In contrast to the prevailing view, the data presented in this study suggest that the major component of the arachidonic acid effect is a direct action on the protein or the protein-lipid boundary caused by the arachidonic acid present in the water phase. The main support for this idea are the data presented in ; the inhibition disappears after dilution of the liposomes, although about 95% of the arachidonic acid is still present in the membranes. Thus, dilution of the water phase does not appreciably affect the lipid phase, but it removes the inhibition. An unspecific effect on the membrane fluidity cannot be the predominant mode of action. This interpretation is supported by the finding (Fig. 3) that esterification of the carboxylic ``head'' of the fatty acid destroys the inhibitory activity in spite of the fact that the esterification increases the lipophilicity and thereby the tendency of the molecules to associate with the membranes. Furthermore, as is shown in Fig. 4, it is possible to increase the sensitivity of the glutamate transporters to freshly added aqueous arachidonic acid by pretreating the liposomes with either arachidonic acid or the inactive ethyl ester. Finally, Fig. 5shows that the ethyl ester strongly potentiates the effect of arachidonic acid. The easiest way to explain these data is to assume that there are two different types of binding sites for arachidonic acid on the liposomes. The first type is sites directly involved in the inhibition of glutamate transport. These sites require aqueous arachidonic acid and have to be on the transporter protein or protein-lipid boundary. The second type is sites in the lipids. These sites act like a sink and remove the fatty acid from the water phase, making it unavailable for interaction with the transporter protein. The ethyl ester probably substitutes for the free acid only at these sites. When these lipidic sites are saturated by the ethyl ester, added arachidonic acid will be more potent because more of it will be available for interaction with the transporter protein.

The substrate affinity of the glutamate transporters has been a matter of discussion (for review, see Danbolt (1994)). We show here that the uptake in proteoliposomes is not linear with time for more than 2 s at highest glutamate concentrations. Because of this, the true Kvalues are at least 10-fold higher than those previously reported for proteoliposomes (Danbolt et al., 1990; Pines et al., 1992; Pines and Kanner, 1990), but they are in agreement with those determined electrophysiologically.

In conclusion, our data suggest that inhibition of glutamate uptake by arachidonic acid derives from the reversible binding of free aqueous arachidonic acid to either the transporter protein or the protein-lipid boundary. The binding, which requires both a hydrophobic cis-polyunsaturated carbon chain and a hydrophilic carboxyl group, is probably of low affinity, since it is easily reversed by simple dilution. Binding of arachidonic acid to the phospholipid membranes does not seem to affect the function of the glutamate transporter. However, the lipids seem to bind arachidonic acid tightly, thereby extracting it from the water phase, i.e. from the active pool. This would imply that the active arachidonic acid concentration is significantly lower than that nominally applied. Since the GABA transporter(s) were inhibited in a similar manner although they belong to a different protein family with no significant primary structure identity with the glutamate transporters (for review, see Danbolt (1994)), the effect of arachidonic acid on membrane proteins in general could be due to the aqueous rather than the lipid bound fatty acid. To our knowledge, this is the first report where it has been possible to conclude whether the fatty acid effect is directly on the target protein or secondary to changes in the membrane fluidity. Direct modulation by arachidonic acid of integral membrane proteins has been reported ( e.g. Ordway et al. (1991)), but these studies were not able to distinguish the two possibilities.

  
Table: Integrity of liposomes loaded with labelled glutamate after exposure to arachidonic acid

Liposomes (without protein) loaded with labeled glutamate and suspended in glutamate free medium were exposed to arachidonic acid (50, 125, or 300 µM) for 15, 30, or 120 min and collected on Millipore filters as described under ``Experimental Procedures.'' The results represent the mean ± S.E. of three experiments expressed as percent of control (values obtained without arachidonic acid). Control values for the experiments with exposure time of 15, 30, and 120 min were 4511 ± 221, 4667 ± 15, and 5371 ± 653 cpm. Addition of Triton X-100 (0.5 µl/ml) for 30 s reduced the radioactive content to less than 1%.


  
Table: Reversibility of arachidonic acid-induced inhibition in liposomes reconstituted with glutamate transporter protein

Liposomes with purified glutamate transporters and internal potassium were suspended in potassium-free PBSG and preincubated (15 min) with 300 µM arachidonic acid (AA).



FOOTNOTES

*
This work was supported by a short term fellowship from the European Science Foundation (to D. T.), Telethon (Grant 586), student research fellowships from the Norwegian Research Council (to K. P. L., and O. G.), and Lars Fylkesakers stiftelse. 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.

§
To whom correspondence should be addressed: Fax: 47 22 85 12 78; E-mail: ncd@pons.uio.no.

The abbreviations used are: GABA, -aminobutyric acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.


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