Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells with glutamate for metabolic purposes. In the kidney, glutamate transporters are involved in reabsorption of filtered acidic amino acids, regulation of ammonia and bicarbonate production, and protection of cells against osmotic stress.
- sodium-dependent transporters
- intracellular pH
- cell volume regulation
- metabolic acidosis
glutamate transporters and structurally related neutral amino acid transporters constitute a distinct family of Na+-dependent transporters. A total of five mammalian isoforms of glutamate transporters, EAAC1 (EAAT3), GLT1 (EAAT2), GLAST (EAAT1), EAAT4, and EAAT5, and two system ASC (alanine, serine, cysteine) amino acid transporters, ASCT1 and ASCT2, have been identified (1, 3, 12, 21, 26,32, 41, 47, 55, 62) (Table 1). All EAAT family members display an almost identical hydrophobic profile. They have eight transmembrane domains and a structure reminiscent of a pore loop between the 7th and 8th domains (15).
Glutamate transporters exhibit a unique coupling pattern to inorganic ions that allows them to efficiently pump glutamate into cells. Transport is electrogenic, and the uptake of one glutamate molecule is coupled to the cotransport of three sodium ions and one proton and the countertransport of one potassium ion, resulting in the net translocation of two positive charges per transport cycle (4, 6, 25,64, 70). Glutamate transport is accompanied by intracellular acidification (22), and transport of glutamate is driven by a pH gradient (39). Mechanistic studies have shown that the transport process is ordered and that sodium and potassium are translocated in distinct steps. The sodium ions bind first, followed by glutamate. H+ either binds directly to the transporter protein (39) or is used to protonate glutamate, which may be translocated in its protonated form (71). After translocation of sodium, proton, and glutamate and their release on the inside of the cell, potassium binds on the inside and is translocated outwards to complete the translocation cycle (22, 24, 42). It has been shown that glutamate transporters exhibit an anion conductance, which is induced by glutamate in a sodium-dependent fashion (12, 63). The physiological role of this anion conductance is not completely understood (see accompanying report of Fairman and Amara, Ref. 11a).
The glutamate transporter GLT-1 has been both purified to near homogeneity and functionally reconstituted by Kanner and colleagues (11, 41). It represents ∼0.6% of the protein of crude synaptosomal fractions and accounts for 90% of all glutamate uptake activity in the brain. GLT-1 has a predominant glial localization (61). Mutations in GLT-1 have been implicated in amyotrophic lateral sclerosis (33). The glutamate transporter GLAST is strongly expressed by Bergmann glia in the cerebellum (55) and more moderately by astrocytes and ependymal cells throughout the central nervous system. It contributes ∼5–10% to the glutamate uptake capacity in brain.
The glutamate transporter EAAC1 has been isolated by expression cloning in Xenopus oocytes (21). It is expressed in neurons throughout the central nervous system, as well as in epithelial tissues (intestine, liver, and kidney) (49).
Expression of the glutamate transporter EAAC1 in rat kidney. In general, filtered solutes such as glucose and amino acids are reabsorbed in proximal tubules by segment-specific low-affinity and high-affinity transporters. For example, the low-affinity Na+-glucose cotransporter SGLT2, which reabsorbs the bulk of the filtered glucose, is located in S1 segments, and the high-affinity Na+-glucose cotransporter SGLT1, which removes the last traces of filtered glucose, is located in S3 segments (16). This arrangement is slightly different for glutamate transporters, because glutamate also has important metabolic functions. Earlier studies revealed that high-affinity Na+- and K+-dependent glutamate transporters are present in both brush-border and basolateral membranes (46) to maintain a relatively high intracellular glutamate concentration.
On the basis of in situ hybridization and immunocytochemistry, the predominant expression of EAAC1 in rat kidney is in the apical membrane of proximal tubule S2 and S3 segments (49). This supports the concept that EAAC1 is the high-affinity low-capacity apical transporter responsible for final reabsorption of glutamate that escaped the early part of the proximal tubule, especially when the filtered load of glutamate is increased (7, 50). EAAC1 expression was also detected in thin descending limbs of long-looped nephrons, medullary thick ascending limbs, and distal convoluted tubules.
There is also evidence for a kidney Na+- and K+-dependent low-affinity glutamate transporter. Microperfusion and free-flow micropuncture studies in rat kidney revealed that the bulk (>90%) of filtered acidic amino acids is reabsorbed within the first third of the proximal convolution (S1 segments) (50, 51). The low-affinity transporter would be expected to account for most of the glutamate reabsorption in early proximal tubule segments, but its molecular identity remains elusive. Likewise, a cDNAs encoding the basolateral high-affinity glutamate transporters has not yet been isolated.
EAAC1 as a metabolic regulator in kidney proximal tubules. Part of the glutamate absorbed via EAAC1 in proximal straight tubules can be assumed to be the product of phosphate-independent glutaminase (PIG) (Fig. 1). Consistent with this concept, the distribution of expression of EAAC1 in the apical membranes of rat proximal tubule S2 and S3 segments well-matches that of PIG. In metabolic acidosis, however, the reduced luminal bicarbonate concentration inhibits PIG (66), resulting in decreased glutamate production and decreased glutamate uptake by EAAC1 (45, 50). The existing intracellular acidosis accelerates glutamate flux through the glutamate dehydrogenase (GDH) (see the accompanying report of Nissim, Ref. 39a), resulting in a further decrease in intracellular glutamate concentration, which in turn activates the phosphate-dependent glutaminase (PDG) flux (14) (see Fig.1). Intracellular acidosis also activates α-ketoglutarate dehydrogenase (34) and phosphoenolpyruvate carboxykinase (PEPCK) (65). Glutamine utilized is coupled to gluconeogenesis and/or CO2 production (5). As a consequence, glutamine, produced during metabolic acidosis in muscle, is utilized by kidney proximal tubules to form two molecules of bicarbonate, which enter the blood through the basolateral bicarbonate cotransporter NBC, as well as two molecules of ammonium, which enter the lumen via the sodium/proton exchanger NHE3 (28, 38). Excretion of ammonium serves to effectively eliminate protons while the bicarbonate replenishes the plasma alkaline reserve during metabolic acidosis. Thus the purpose of the production of bicarbonate and ammonium is to counteract the reduced plasma pH during metabolic acidosis. The combined action of PIG and EAAC1 offers a sensing mechanism for metabolic acidosis, which, in concert with cellular acidosis, regulates acid base homeostasis through acceleration of the catabolism of glutamine in mitochondria followed by production of bicarbonate and ammonium.
Whether EAAC1 is regulated in the kidney in response to metabolic acidosis has not been demonstrated. Metabolic acidosis increased glutamate uptake in LLC-PK1-F+cells (37), but this might be in part explained by the direct effect of lower extracellular pH on transport activity, since glutamate transport is coupled to the cotransport of H+ (22) (see above).
Functional role of EAAC1 in the distal tubule segments in kidney. Expression of EAAC1 in segments other than proximal tubules raises the question of whether glutamate might be used as a metabolic fuel in these segments. Previous studies on the specificity of metabolic fuels that are required for each nephron segment to maintain cellular ATP levels revealed that glutamate can be metabolized and oxidized to produce CO2 in thick ascending limbs (29). Glutamate can enter the tricarboxylic acid cycle in an anaplerotic reaction after transamination with pyruvate to form alanine and α-ketoglutarate, which subsequently serve as fuels for the citric acid cycle. However, glutamate is probably not the preferred substrate with which to maintain cellular ATP content in thick ascending limbs and to energize transepithelial Na+ transport (8, 67), given the high metabolic expenditure required to absorb glutamate: coupling to the cotransport of three Na+ ions and the countertransport of one K+ion.
Possible involvement of EAAC1 in cell volume regulation. Expression of EAAC1 in thin descending limbs of long-looped nephron suggests a potential role for acidic amino acids in cell volume regulation in the medullary interstitium (56). Although there is no evidence that the concentration of glutamate or aspartate alone is sufficiently high to account for the increased osmolality inside the cell, the sum of these acidic amino acids and others may allow their contribution as significant organic osmolytes in cells of the renal medulla. Glutamate and aspartate may balance both pH changes and osmotic pressure simultaneously. Both amino acids were shown to be accumulated in the medullary interstitium (35). EAAC1 may allow entry of glutamate to regulate cell volume in response to osmotic stress. Recently, it has been shown that hypertonic stress results in an increase in EAAC1 expression (9, 13) (see the accompanying report from McGivan and Nicholson, Ref. 35a). These results imply that there must be a specific mechanism for glutamate efflux as part of the hyposmotic regulatory volume response. Because of the coupling pattern of EAAC1, which includes countertransport of K+, it is unlikely that the efflux is due to the reversed operation of the transporter. In astrocytes, swelling-activated channels have been identified through which anionic osmolytes can be released from cells. Astrocytic swelling occurs in ischemia and traumatic brain injury as part of the edema response and was shown to cause both inhibition of glutamate uptake and an increase in its release (27). Hyposmotic glutamate release was also demonstrated during volume regulation by cardiac cells (53). Thus the current concept is that there are anion-permeant channels that can dump glutamate in response to hyposmolality (56). However, a cDNA encoding such a channel has not yet been isolated.
It is furthermore conceivable that epithelial cells in distal tubule segments accumulate glutamate not only as an osmolyte but as a regulator of the hypertonic response. In the acute hypertonic condition, PIG is downregulated (T. Welbourne, personal communication), and less glutamate may be taken up by EAAC1 as a the result of decreased extracellular availability. This results in an initial reduction in intracellular glutamate levels, increased PDG activity, and accelerated glutamine uptake via system A or ASC, which leads to increased glutamate production. However, in contrast to proximal tubules where glutamate is converted to α-ketoglutarate by GDH for catabolic purposes (see Fig. 1), GDH levels are relatively low in distal segments (68) and glutamate may be transaminated to alanine. Thus decreased uptake of glutamate by EAAC1 may trigger intracellular accumulation of glutamate and alanine as volume-regulating osmolytes. Since system A in the basolateral membrane is highly stimulated in cells during hypertonic stress (18, 44), this concept would provide an explanation for the high expression of PDG in the distal nephron (10).
Dicarboxylic aminoaciduria.Dicarboxylic aminoaciduria is a rare disorder in which glutamate and aspartate clearances exceed glomerular filtration rates (20, 36, 57,60). Furthermore, neurological abnormalities may be associated with the disease (36). Recent studies of EAAC1 (−/−) knockout mice revealed that loss of EAAC1 function results in dicarboxylic aminoaciduria, in addition to some behavioral abnormalities (40) and seizures (43). However, whether patients with dicarboxylic aminoaciduria have a defect in EAAC1 still remains to be elucidated.
Address for reprint requests and other correspondence: M. A. Hediger, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Rm. 570, Boston, MA 02115 (E-mail:).
This article is the second of five in this forum, which is based on a series of reports on glutamate transport and glutamate metabolism that was first presented at Experimental Biology ’98 in San Francisco, CA.
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