Modulation of Human Glutamate Transporter Activity by Phorbol Ester

Abstract: Termination of synaptic glutamate transmission depends on rapid removal of glutamate by neuronal and glial high-affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least five subtypes of Na⁺-dependent mammalian glutamate transporters exist. Our study demonstrates that Y-79 human retinoblastoma cells express a single Na⁺-dependent glutamate uptake system with a K _m of 1.7 ± 0.42 µ M that is inhibited by dihydrokainate and dl - threo -β-hydroxyaspartate (IC₅₀ = 0.29 ± 0.17 µ M and 2.0 ± 0.43 µ M , respectively). The protein kinase C activator phorbol 12-myristate 13-acetate caused a concentration-dependent inhibition of glutamate uptake (IC₅₀ = 0.56 ± 0.05 n M ), but did not affect Na⁺-dependent glycine uptake significantly. This inhibition of glutamate uptake resulted from a fivefold decrease in the transporter's affinity for glutamate, without significantly altering the V _max. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate protein kinase C, did not alter glutamate uptake significantly. The phorbol 12-myristate 13-acetate-induced inhibition of glutamate uptake was reversed by preincubation with staurosporine. The biophysical and pharmacological profile of the human glutamate transporter expressed by the Y-79 cell line indicates that it belongs to the dihydrokainate-sensitive EAAT2/GLT-1 subtype. This conclusion was confirmed by western blot analysis. Protein kinase C modulation of glutamate transporter activity may represent a mechanism to modulate extracellular glutamate and shape postsynaptic responses.     

Variant Forms of Neuronal Glutamate Transporter Sites in Alzheimer's Disease Cerebral Cortex

Abstract: The displacement of Na⁺-dependent d -[³H]-aspartate binding by unlabeled d -aspartate or the inhibitors dl - threo -β-hydroxyaspartate, l -cysteate, l -glutamate, dihydrokainate, dl -α-aminoadipate, α-methyl- dl -glutamate, and 1-aminocyclobutane- cis -1,3-dicarboxylate was used to characterize the high-affinity glutamate/aspartate uptake site in human cerebral cortex. Synaptosomal membranes were prepared from tissue obtained at autopsy from nondemented control, Alzheimer's disease (AD), and diffuse Lewy body disease (DLBD) cases. Areas that are damaged in AD (midtemporal, frontal, caudal cingulate, and hippocampal cortices) were compared with those that are spared (occipital and motor cortices). Profiles of the affinities ( K _a values) of the ligands showed that areas spared from damage in AD cases differed significantly from equivalent areas in control ( p < 0.001) and DLBD ( p < 0.001) cases and also from areas susceptible to damage in the same AD cases ( p < 0.001). Areas susceptible to damage in AD showed comparable profiles across the three case groups ( p = 0.980). The glutamate/aspartate uptake site may be regionally variant in AD cases, and this may underlie local excitotoxicity. d -[³H]Aspartate binding site density was significantly lower in both dementia groups (control vs. AD, p < 0.001; control vs. DLBD, p = 0.009; but AD vs. DLBD, p = 0.528); within-group differences were not significant (control, p = 0.874; AD, p = 0.285; DLBD, p = 0.741).     

l-Aspartate as an amino acid neurotransmitter: mechanisms of the depolarization-induced release from cerebrocortical synaptosomes

The role of l -aspartate as a classical neurotransmitter of the CNS has been a matter of great debate. In this study, we have characterized the main mechanisms of its depolarization-induced release from rat purified cerebrocortical synaptosomes in superfusion and compared them with those of the well-known excitatory neurotransmitter l -glutamate. High KCl and 4-aminopyridine were used as depolarizing agents. At 15 mM KCl, the overflows of both transmitters were almost completely dependent on external Ca²⁺. At 35 and 50 mM KCl, the overflows of l -aspartate, but not those of l -glutamate, became sensitive to dl -threo-β-benzyloxyaspartic acid ( dl -TBOA), an excitatory amino acid transporter inhibitor. In the presence of dl -TBOA, the 50 mM KCl-evoked release of l -aspartate was still largely external Ca²⁺-dependent. The dl -TBOA insensitive, external Ca²⁺-independent component of the 50 mM KCl-evoked overflows of l -aspartate and l -glutamate was significantly decreased by the mitochondrial Na⁺/Ca²⁺ exchanger blocker CGP 37157. The Ca²⁺-dependent, KCl-evoked overflows of l -aspartate and l -glutamate were diminished by botulinum neurotoxin C, although to a significantly different extent. The 4-aminopyridine-induced l -aspartate and l -glutamate release was completely external Ca²⁺-dependent and never affected by dl -TBOA. Superimposable results have been obtained by pre-labeling synaptosomes with [³H] d -aspartate and [³H] l -glutamate. Therefore, our data showing that l -aspartate is released from nerve terminals by calcium-dependent, exocytotic mechanisms support the neurotransmitter role of this amino acid.     

l-Aspartate but Not the d Form Is Secreted Through Microvesicle-Mediated Exocytosis and Is Sequestered Through Na+-Dependent Transporter in Rat Pinealocytes

Abstract: Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca²⁺ ionophore) in the presence of Ca²⁺ and was proportional to the amount of l -glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca²⁺ channel blockers. Bay K 8644, an agonist for the L-type Ca²⁺ channel, stimulated the release of aspartate 2.1-fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the l form, although pinealocytes also contain the d form in their cytoplasm, corresponding to ～30% of the total free aspartate. Radiolabeled l -aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na⁺-dependent manner. The Na⁺-dependent uptake of l -aspartate was strongly inhibited by l -cysteine sulfinate, β-hydroxyaspartate, and l -serine-O-sulfate, inhibitors for the Na⁺-dependent glutamate/aspartate transporter on the plasma membrane. Na⁺-dependent sequestration of l -aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that l -aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na⁺-dependent transporter at the plasma membrane. The possible role of l -aspartate as an intercellular chemical transmitter in the pineal gland is discussed.     

Glutamate Uptake Stimulates Na+,K+-ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na⁺-dependent transporter. Evidence had been provided that Na⁺,K⁺-ATPase might be involved in this process. We have now measured the activity of Na⁺,K⁺-ATPase in cultured astrocytes, using ouabain-sensitive ⁸⁶Rb uptake as an index. l -Glutamate increases glial Na⁺,K⁺-ATPase activity in a concentration-dependent manner with an EC₅₀ = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K _0.5 = 113 µ M ), compatible with the presence of an α₁ isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K _0.5 = 20 n M ), thus revealing a high-affinity site akin to the α₂ isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na⁺,K⁺-ATPase that can be mobilized in response to increased neuronal activity.     

STIMULATORY EFFECT OF l-GLUTAMATE AND RELATED AMINO ACIDS ON [3H]DOPAMINE RELEASE FROM RAT STRIATUM: AN IN VITRO MODEL FOR GLUTAMATE ACTIONS

Abstract— The effects of l -glutamate and a number of structural analogues on the spontaneous release of [³H]dopamine from slices of rat striatum were examined. Glutamate, and other excitatory amino acids produced a marked stimulation of [³H]DA release which was Ca²⁺-dependent and unaffected by either procaine or tetrodotoxin. The glutamate-stimulated release was abolished in kainate-lesioned striatum. The action of glutamate was effectively antagonised by glutamamate diethylester and 2-amino-4-phosphonobutyric acid, but only weakly by l -methionine- dl -sulfoximine. Other proposed amino acid antagonists were inactive. The likely site of the releasing action of l -glutamate on presynaptic sites on nigro-striatal DA terminals is discussed.     

Identification of a System N-Like Na+-Dependent Glutamine Transport Activity in Rat Brain Neurons

Abstract: Glutamine is a primary precursor for the biosynthesis of the neurotransmitters glutamate and γ-aminobutyric acid. It is proposed that glutamine, synthesized and released by astrocytes, is transported into the neuron for subsequent conversion to neurotransmitters. To provide a more complete characterization of this process, we have delineated the transport systems for glutamine uptake in primary cultures of brain neuronal cells from 1-day-old rats. The Na⁺-dependent glutamine entry is mediated by system A, system ASC, and a third, previously unidentified, activity that has been tentatively designated as system N^b. System N^b activity can be monitored by assaying Na⁺-dependent [³H]glutamine uptake in the presence of 2 m M concentrations of both 2-(methylamino)isobutyric acid and threonine to block uptake by systems A and ASC, respectively. The newly identified transport activity exhibits an apparent substrate specificity that is unique compared with the hepatic system N, because it is inhibited by glutamine and asparagine, but not by histidine. Also, the affinity of system N^b for glutamine, as estimated from K _m values, is significantly greater than that observed for the hepatic and muscle Na⁺-dependent glutamine transporters, systems N and N^m. In sharp contrast to the hepatic system N transporter, system N^b exhibits a relative insensitivity to pH and does not permit Li⁺ substitution for Na⁺ as the cosubstrate. The substrate specificity, kinetic analysis, pH sensitivity, and cation dependence of this transport activity indicate that it represents a glutamine transport system not previously identified.     

Increase in Chloride-Dependent l-Glutamate Transport Activity in Synaptic Membrane After In Vitro Ischemic Treatment

Abstract: The effect of energy failure on Cl⁻-dependent l -glutamate ( l -Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose-deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl⁻-dependent l -[³H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased l -[³H]Glu uptake was inhibited by l -Glu, dl -2-amino-4-phosphonobutyrate, l -homocysteic acid, l -cystine, 4,4'-diisothiocyano-2,2'-disulfonic stilbene, and removal of Cl⁻. Uptakes of Na⁺-dependent l -[³H]-Glu, [³H]GABA, and [³H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the V _max value without affecting the K _m value. The increased l -[³H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose-containing solution. ATP content in brain slices decreased to <10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose-containing solution. Treatment with veratrine, 2,4-dinitrophenol, carbonyl cyanide m -chlorophenylhydrazone, and NaCN in normoxic conditions increased l -[³H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl⁻-dependent l -Glu transport activity in synaptic membranes increases in ischemia- or hypoxia-induced brain energy failures.     

N-System Amino Acid Transport at the Blood-CSF Barrier

Abstract: Despite l -glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in l -[¹⁴C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na⁺-dependent mechanism. The Na⁺-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na⁺-dependent uptake was inhibited neither by the A-system substrate α-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and l -glutamate γ-hydroxamate, three N-system substrates. Replacement of Na⁺ with Li⁺ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The V _max and K _max for glutamine transport by this system were 8.1 ± 0.3 nmol/mg/min and 3.3 ± 0.4 m M , respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Regional Deafferentiation Down-Regulates Subtypes of Glutamate Transporter Proteins

Abstract: Low extracellular glutamate content is maintained primarily by high-affinity sodium-dependent glutamate transport. Three glutamate transporter proteins have been cloned: GLT-1 and GLAST are astroglial, whereas EAAC1 is neuronal. The effects of axotomy on glutamate transporter expression was evaluated in adult rats following unilateral fimbria-fornix and corticostriatal lesions. The hippocampus and striatum were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted using antibodies directed against GLT-1, GLAST, EAAC1, and glial fibrillary acidic protein and assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 14 days postlesion. GLAST immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 7 and 14 days postlesion. No alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 14 days postlesion within the ipsilateral hippocampus and at 7 and 14 days postlesion within the ipsilateral striatum. By 30 days postlesion, glutamate transporters and d -[³H]aspartate binding returned to control levels. This study demonstrates the down-regulation of primarily glial, and not neuronal, glutamate transporters following regional disconnection.     

Oxidative Stress, Hypoxia, and Ischemia-Like Conditions Increase the Release of Endogenous Amino Acids by Distinct Mechanisms in Cultured Retinal Cells

Abstract: The aim of this study was to elucidate the mechanisms by which retinal cells release endogenous amino acids in response to ascorbate/Fe²⁺-induced oxidative stress, as compared with chemical hypoxia or ischemia. In the absence of stimulation, oxidative stress increased the release of aspartate, glutamate, taurine, and GABA only when Ca²⁺ was present. Under hypoxia or ischemia, the release of aspartate, glutamate, glycine, alanine, taurine, and GABA increased mainly by a Ca²⁺-independent mechanism. The increased release observed in N -methyl- d -glucamine⁺ medium suggested the reversal of the Na⁺-dependent amino acid transporters. Upon oxidative stress, the release of aspartate, glutamate, and GABA, occurring through the reversal of the Na⁺-dependent transporters, was reduced by about 30%, although the release of taurine was enhanced. An increased release of [³H]arachidonic acid and free radicals seems to affect the Na⁺-dependent transporters for glutamate and GABA in oxidized cells. All cell treatments increased [Ca²⁺]_i (1.5 to twofold), although no differences were observed in membrane depolarization. The energy charge of cells submitted to hypoxia or oxidative stress was not changed. However, ischemia highly potentiated the reduction of the energy charge, as compared with hypoglycemia or hypoxia alone. The present work is important for understanding the mechanisms of amino acid release that occur in vivo upon oxidative stress, hypoxia, or ischemia, frequently associated with the impairment of energy metabolism.     

Endothelin Evokes Efflux of Glutamate in Cultures of Rat Astrocytes

Abstract: Excessive release of glutamate, from glial cells as well as neurons, is thought to be a major cause of neuronal death in ischemia. To investigate glutamate release from glial cells, we measured glutamate efflux from cultures of rat astrocytes preloaded with l -[³H]-glutamate. Glutamate efflux was induced by either 60 m M KCl or Na⁺-free medium, suggesting that the efflux is due to the reversed operation of a Na⁺- and K⁺-coupled glutamate uptake machinery. While investigating various neuropeptides and neurotransmitters, we found that endothelin (ET) specifically induced efflux of glutamate. Northern blot analysis and binding study showed that the ET type B receptor (ET_B-R) subtype was expressed two to three times more densely than the ET type A receptor (ET_A-R) in astrocytes. The ET_B-R antagonist IRL 2500 partially inhibited efflux of glutamate induced by 1 n M ET-1 in a concentration-dependent manner, causing a maximal inhibition of 60% at 1 µ M . However, the ET_A-R antagonist BQ-123 did not cause significant inhibition even at 10 µ M . Combination of both antagonists completely inhibited the ET-1-induced efflux. These results indicate that both receptor subtypes are involved in efflux of glutamate with a major contribution from the ET_B-R. Our findings suggest that ET, which is known to be released in ischemia, may exacerbate neurodegeneration by stimulating efflux of glutamate.     

Alterations in Glutamate Transporter Protein Levels in Kindling-Induced Epilepsy

Abstract: There is increasing evidence that levels of glutamate are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. Modulation of high-affinity glutamate uptake is a potential mechanism responsible for the elevated levels observed with seizures. To date, three distinct Na⁺-dependent glutamate transporters have been cloned from rat and rabbit: GLT-1, GLAST, and EAAC-1. We performed a series of experiments to determine whether levels of these transporters are altered in amygdala-kindled rats. Levels of GLT-1, GLAST, and EAAC-1 were examined in three brain regions (hippocampus, piriform cortex/amygdala, and limbic forebrain) by quantitative immunoblotting using subtype-specific antibodies. GLAST protein was down-regulated in the piriform cortex/amygdala region of kindled rats as early as 24 h after one stage 3 seizure and persisting through multiple stage 5 seizures. In contrast, kindling induced an increase in EAAC-1 levels in piriform cortex/amygdala and hippocampus once the animals had reached the stage 5 level. No changes in GLT-1 were observed in any region examined. Changes in transporter levels could contribute to the changes in glutamate levels seen with kindling.     

Taurine Interactions with Chick Retinal Membranes

Abstract: Binding of [³H]taurine to whole retinal membranes and to membranes obtained from retinal subcellular fractions was studied. [³H]Taurine bound to chick retinal membranes with high affinity and specificity. Two types of [³H]taurine binding associated to retinal membranes were observed, one with a K_D= 0.68 μM and the other one with a K_D,= 9.32 μM. Both types of binding were highly Na⁻-dependent. The Na⁺-dependent taurine binding was antagonized by strychnine. Bound [³H]taurine was effectively displaced by β-alanine but not by GABA or glycine. Taurine binding was preferentially localized in membranes obtained from the crude synaptosomal fraction, although it is also present in substantial amounts in all retinal membranes. A Na⁺-independent [³H]taurine binding exhibiting properties which might represent interaction with postsynaptic receptor sites could not be demonstrated in the chick retina.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.     

Cationic and Anionic Requirements for the Binding of 2β-Carbomethoxy-3β-(4-Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier

Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [³H]CFT binding to synaptosomal P₂ membranes requires low concentrations of Na⁺ (peak binding between 20 and 50 m M Na⁺), is stimulated by phosphate anion or l^-, but is unaffected or only slightly affected by F^-, Cl^-, Br^-, NO₃^-, or SO₄^2-, Concentrations of Na⁺ of >50 m M become inhibitory except in the presence of l^-, which shifts peak binding levels toward higher Na⁺ concentrations and also elevates the peak binding level. K⁺ strongly decreased [³H]CFT binding with a shallow inhibition curve, and Na⁺ could not overcome this effect. Saturation analysis of [³H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 m M ; 1 mM plus 49 mM NaCIversus 10 m M plus 40 m M NaCI; or 1 mM plus 129 m M Nal versus 10 m M plus 120 m M Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.