High-affinity Transport Process Research Articles

Universal fluorescent sensors of high-affinity iron transport, applied to ESKAPE pathogens

Sensitive assays of biochemical specificity, affinity, and capacity are valuable both for basic research and drug discovery. We created fluorescent sensors that monitor high-affinity binding reactions and used them to study iron acquisition by ESKAPE bacteria, which are frequently responsible for antibiotic-resistant infections. By introducing site-directed Cys residues in bacterial iron transporters and modifying them with maleimide fluorophores, we generated living cells or purified proteins that bind but do not transport target compounds. These constructs sensitively detected ligand concentrations in solution, enabling accurate, real-time spectroscopic analysis of membrane transport by other cells. We assessed the efficacy of these "fluorescent decoy" (FD) sensors by characterizing active iron transport in the ESKAPE bacteria. The FD sensors monitored uptake of both ferric siderophores and hemin by the pathogens. An FD sensor for a particular ligand was universally effective in observing the uptake of that compound by all organisms we tested. We adapted the FD sensors to microtiter format, where they allow high-throughput screens for chemicals that block iron uptake, without genetic manipulations of the virulent target organisms. Hence, screening assays with FD sensors facilitate studies of mechanistic biochemistry, as well as discovery of chemicals that inhibit prokaryotic membrane transport. With appropriate design, FD sensors are potentially applicable to any pro- or eukaryotic high-affinity ligand transport process.

Transport of gabapentin by LAT1 (SLC7A5)

Gabapentin is used in the treatment of epilepsy and neuropathic pain. Gabapentin has high and saturable permeability across the BBB, but no mechanistic studies underpinning this process have been reported. The aim of the current study was to investigate the transport of gabapentin in a model of the BBB, identify the important drug transporter(s) and to use mathematical modelling to quantify the processes involved. A human brain endothelial cell line (hCMEC/D3) was utilised as an in-vitro model of the BBB. Uptake of radiolabeled gabapentin into cells in the presence of chemical inhibitors, siRNA or overexpressed drug transporters of interest was investigated. Gabapentin was demonstrated to be a LAT1 substrate in brain endothelial cells (LAT1-process; Km=530μM and Vmax=7039pmoles/million cells/min versus other-processes; Km=923μM and Vmax=3656pmoles/million cells/min) and in transfected HEK 293 LAT1 cells (LAT1-process; Km=217μM and Vmax=5192pmoles/million cells/min versus otherprocesses; Km=1546μM and Vmax=3375pmoles/million cells/min). At physiological concentrations of gabapentin, LAT1 mediated transport was 3 or ~10-fold higher than the other transport processes in the two systems, respectively, demonstrating clear selectivity for gabapentin. In-silico structural homology modelling confirmed that LAT1 could have the LeuT conserved fold and functions by the alternative access mechanism. Mathematical modelling of this mechanism revealed revised significance of Vmax and Km so that a low Km may not necessarily imply a high affinity transport process. Gabapentin was negative for OCT like transport and LAT2 activity in the hCMEC/D3 and OCT1 transfected cells. Our data shows that gabapentin is a substrate for the influx transporter LAT1 at therapeutic concentrations.

Cadmium transport in isolated enterocytes of freshwater rainbow trout: Interactions with zinc and iron, effects of complexation with cysteine, and an ATPase-coupled efflux

The present study investigated the mechanisms of intestinal cadmium (Cd) uptake and efflux, using isolated enterocytes of freshwater rainbow trout (Oncorhynchus mykiss) as the experimental model. The apical uptake of free Cd2+ in the enterocytes was a saturable and high-affinity transport process. Both zinc (Zn2+) and iron (Fe2+) inhibited cellular Cd2+ uptake through a competitive interaction, suggesting that Cd2+ enters enterocytes via both Zn2+ (e.g., ZIP8) and Fe2+ (e.g., DMT1) transport pathways. Cellular Cd2+ uptake increased in the presence of HCO3−, which resembled the function of mammalian ZIP8. Cellular Cd2+ uptake was unaffected by Ca2+, indicating that Cd2+ does not compete with Ca2+ for apical uptake. Interestingly, Cd uptake was influenced by the presence of l-cysteine, and under the exposure condition where Cd(Cys)+ was the predominant Cd species, cellular Cd uptake rate increased with the increased concentration of Cd(Cys)+. The kinetic analysis indicated that the uptake of Cd(Cys)+ occurs through a low capacity transport mechanism relative to that of free Cd2+. In addition, Cd efflux from the enterocytes decreased in the presence of an ATPase inhibitor (orthovanadate), suggesting the existence of an ATPase-coupled extrusion process. Overall, our findings provide new mechanistic insights into the intestinal Cd transport in freshwater fish.

Hypertonicity Enhances GABA Uptake by Cultured Rat Retinal Capillary Endothelial Cells

We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [³H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [³H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [³H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.

EAAC1 is expressed in rat and human prostate epithelial cells; functions as a high-affinity L-aspartate transporter; and is regulated by prolactin and testosterone

BackgroundProstate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells.ResultsThe studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells.ConclusionThis study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter.

Transport and intracellular accumulation of vitamin C in endothelial cells: relevance to collagen synthesis

Endothelial cells preserve vascular integrity in part by synthesizing type IV collagen for the basement membrane of blood vessels. Vitamin C, which at physiologic pH is largely the ascorbate mono-anion, both protects these cells from oxidant stress and is required for collagen synthesis. Therefore, cultured endothelial cells were used to correlate intracellular concentrations of ascorbate with its uptake and ability to stimulate collagen release into the culture medium. The kinetics and inhibitor specificity of ascorbate transport into EA.hy926 endothelial cells were similar to those observed in other cell types, indicative of a specific high affinity transport process. Further, transport of the vitamin generated intracellular ascorbate concentrations that were 80–100-fold higher than concentrations in the medium following overnight culture, and transport inhibition with sulfinpyrazone and phloretin partially prevented such ascorbate accumulation. On the other hand, low millimolar intracellular concentrations of ascorbate impaired its transport measured after overnight culture. Synthesis and release of type IV collagen into the culture medium was markedly stimulated by ascorbate in a time-dependent manner, and was saturable with increasing medium concentrations of the vitamin. Optimal rates of collagen synthesis required intracellular concentrations of the vitamin up to 2 mM. Since such concentrations can only be generated by the ascorbate transporter, these results show the necessity of transport for this crucial function of the vitamin in endothelium.

Hemicholinium-3 mustard reveals two populations of cycling choline cotransporters in Limulus

Cholinergic neurons have both a low-affinity and a high-affinity choline transport process. The high-affinity choline transport is sodium dependent and thus it can be referred to as choline cotransport. Choline cotransport has been shown to be up-regulated by neuronal activity. Protein kinase C has also been shown to regulate choline cotransport. Both forms of regulation appear to modulate transport by altering the numbers of choline cotransporters in the nerve terminal membrane. The present study centers on choline cotransporter trafficking in Limulus brain hemi-slice preparations. The competitive, reversible, non-permeant ligand, [ 3H]hemicholinium-3, was used in binding studies to estimate the relative number of choline cotransporters in plasma membranes. The hemicholinium-3 mustard derivative has been shown to be an irreversible, highly selective, non-permeant ligand for the choline cotransporter, and was also used. Hemicholinium-3 mustard binding to the choline cotransporter blocked [ 3H]choline transport and [ 3H]hemicholinium-3 binding. Antecedent elevated potassium exposure of cholinergic tissues has been shown to up-regulate choline transport by the recruitment of additional choline cotransporters to surface membranes. This treatment was also effective in the recruitment of cotransporters following maximal inhibition by hemicholinium-3 mustard of brain hemi-slices. Long-term washout of hemicholinium-3 mustard in hemi-slices resulted in a time-dependent restoration of choline cotransport. Full recovery occurred within 2 h. In uninhibited slice preparations, both staurosporine and chelerythrine, protein kinase C inhibitors, stimulated choline uptake. However, within a 1-h washout recovery of uptake following hemicholinium-3 mustard inhibition, the staurosporine responsive but not chelerythrine responsive transport had returned. On the basis of these findings, we hypothesize the existence of two distinct populations of cycling choline cotransporters, which includes inactive or “silent” transporters.

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells.

The reabsorption of filtered di- and tripeptides as well as certain peptide mimetics from the tubular lumen into renal epithelial cells is mediated by an H+-coupled high-affinity transport process. Here we demonstrate for the first time H+-coupled uptake of dipeptides into the renal proximal tubule cell line LLC-PK1. Transport was assessed 1) by uptake studies using the radiolabeled dipeptide D-[3H]Phe-L-Ala, 2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and 3) by measurement of intracellular pH (pHi) changes as a consequence of H+-coupled dipeptide transport. Uptake of D-Phe-L-Ala increased linearly over 11 days postconfluency and showed all the characteristics of the kidney cortex high-affinity peptide transporter, e.g., a pH optimum for transport of D-Phe-L-Ala of 6.0, an apparent Km value for influx of 25.8 +/- 3. 6 microM, and affinities of differently charged dipeptides or the beta-lactam antibiotic cefadroxil to the binding site in the range of 20-80 microM. pHi measurements established the peptide transporter to induce pronounced intracellular acidification in LLC-PK1 cells and confirm its postulated role as a cellular acid loader.

Uptake of inorganic carbon in the cyanobacterium Synechocystis PCC6803: physiological and genetic evidence for a high-affinity uptake system.

Synechocystis PCC6803 displays two inorganic carbon-uptake processes, a low-affinity one (apparent Km: 300-400 microM) functional in cells grown under standard or limiting inorganic carbon concentrations, and one with a higher affinity (60 +/- 12 microM), detected only in cells adapted to limiting inorganic carbon conditions. A mutational and screening procedure allowed the isolation of a mutant deficient in the high-affinity system, but only slightly impaired in its growth capacities. The mutated genomic region revealed two open reading frames (ORFs), possibly belonging to an operonic structure. A clone in which the downstream ORF, hatR (high-affinity transport), had been inactivated showed a phenotype close to that of the original mutant. Inactivation of the other ORF, hatA, yielded a clone unable to grow in limiting inorganic carbon conditions. The deduced HatA protein showed no homology with any registered protein. It possessed three hydrophobic domains, including a putative signal peptide. Several hypotheses are considered as to its role. The deduced HatR protein, which possessed the features characteristic of the response regulators of the two-component regulatory systems ubiquitous in bacteria, might be a regulator controlling the activity of the high-affinity transport process. It would belong to the subclass of these molecules lacking the DNA-binding domain.

Spodoptera frugiperda (Sf9) cells express novel purine- and pyrimidine-selective nucleoside transporters

Nucleoside fluxes in mammalian cells are mediated by a family of plasma membrane transporters that function by equilibrative or concentrative mechanisms. Members of this family can be identified on the basis of their permeant selectivities and sensitivities to various inhibitors. This study was initiated to characterize nucleoside transport processes of cultured armyworm ovary (Sf9) cells in anticipation of using Sf9 cells for the functional expression of recombinant mammalian nucleoside transporter proteins. Kinetic analysis of zero- trans influx of 3H-nucleosides in Sf9 cells revealed the presence of high ( K m; 10–40 μM) and low ( K m; ⩾0.4 mM) affinity transport processes. Influx of [ 3H]adenosine at 1 μM (high-affinity process) was inhibited only by purine nucleosides (adenosine > 3′-deoxyadenosine > formycin B > guanosine > inosine), and that of [ 3H]uridine was inhibited only by pyrimidine nucleosides (uridine > 3′-deoxyuridine > thymidine). By contrast, fluxes of [ 3H]adenosine and [ 3H]uridine at 100 μM (low-affinity process) were inhibited by both pyrimidine and purine nucleosides. These results suggested the presence of at least two high-affinity transport processes with selectivity for either purine or pyrimidine nucleosides and a low-affinity process with selectivity for both. None of the transport processes were sodium-dependent since uptake of adenosine and uridine was unaffected by elimination of the sodium gradient, and assays that employed formycin B, a non-metabolized permeant of the high-affinity purine-selecive nucleoside transport process, revealed that transport was equilibrative. Nitrobenzylthioinosine, dipyridamole, and dilazep, potent inhibitors of equilibrative nucleoside transport processes in mammalian cells, were poor inhibitors of adenosine and uridine fluxes in Sf9 cells.

Acquisition and storage of inorganic iron and hemin by the yersiniae

Pathogenic Yersinia express a complex array of iron-regulated functions and possess mechanisms for inorganic iron and hemin acquisition. These include a unique temperature-regulated hemin storage system, high-affinity transport processes for hemin and inorganic iron, and an iron-responsive regulatory system controlling gene expression. The genetic organization of these systems illuminates several aspects of iron metabolism in the yersiniae.

Characterization of a Mg-Dependent, Na-Inositol Co-Transport Process in Cardiac Sarcolemmal Vesicles

[ 3H]- myo-Inositol transport into cardiac sarcolemmal vesicles is both a time- and Na +-dependent process. Transport was stimulated 3-fold by 1 μM valinomycin suggesting the process is electrogenic. myo-Inositol transport was dependent upon the presence of Mn 2+ or Mg 2+ but not Ca 2+. Kinetic analysis revealed a high affinity transport process that was saturable (V max, 650 ± 71 pmoles myo-inositol/mg protein/min; K m, 37.7 ± 1.3 μM) and a low affinity process that was unsaturable up to 1.0 mM substrate. Transport was not inhibited by 1.0 mM of either L-glucose, D- or L-galactose. However, D-glucose and L-fucose at 1.0 mM were inhibitory. Higher concentrations (30 mM) of each of the sugars inhibited transport. myo -Inositol transport was also inhibited by the inositol isomers (1.0 mM), D- chiro-inositol, epi-inositol and scyllo-inositol with scyllo-inositol being most effective. Kinetic analysis established sycllo -inisitol as a competitive inhibitor of cardiac myo -inositol transport, increasing the K m for substrate three-fold (122 ± 21 μM). These data indicate that inositol transport across cardiac sarcolemma is a Mg 2+-dependent Na + co-transport process that is electrogenic and stereospecific.

O-187 - Inhibitory effect of dilazep, an adenosine potentiator, on high-affinity transport process of adenosine in cultured vascular endothelial cells

O-187 - Inhibitory effect of dilazep, an adenosine potentiator, on high-affinity transport process of adenosine in cultured vascular endothelial cells

Inositol Metabolism During Neuroblastoma B50 Cell Differentiation: Effects of Differentiating Agents on Inositol Uptake

Inositol uptake was studied in the rat CNS neuroblastoma B50 cell line. Eadie-Hofstee analysis of the uptake pattern reveals two defined modes of inositol entry into the cell. The high-affinity uptake component requires the presence of extracellular sodium and is inhibited by phloridzin. Analysis of the uptake velocities of the high-affinity uptake component provided the following apparent kinetic parameters: Km = 13.7 microM and Vmax = 14.7 pmol/mg of protein/min (without correcting for residual diffusion) and Km = 12.9 microM and Vmax = 12.3 pmol/mg of protein/min (with correction). At physiological concentrations, the high-affinity transport process contributes approximately 70% to total uptake; the remainder is due to a low-affinity diffusion-like process. Uptake inhibition studies reveal that the uptake process is sensitive to ouabain, amiloride, and dichlorobenzamil inhibition but relatively insensitive to cytochalasin B or phloretin. When neuroblastoma B50 cells are induced to differentiate morphologically with high extracellular calcium or with dibutyryl cyclic AMP, a significant decrease in inositol uptake is observed. The dibutyryl cyclic AMP-mediated inhibition of uptake affects only the high-affinity uptake component and is noncompetitive in nature. The high extracellular calcium-mediated inhibition is less specific; it involves "disappearance" of the high-affinity process, some inhibition of the low-affinity process, and an increase of inositol efflux. The significance of these observations is discussed in the context of neuroblastoma B50 cell differentiation.

What constitutes a serotonergic neurone in the retina?

When exposed to exogenous serotonin, specific neurones in retinas from different species (guinea pig, rabbit, frog, pigeon and goldfish) take the amine up; these same neurones do not reveal endogenous serotonin following immunocytochemistry for the localization of the amine. The use of autoradiography and immunocytochemistry to localize the uptake of radioactive or unlabelled amine gives identical results, with the exception of the guinea pig retina, where a subset of horizontal-like cells specifically takes up a metabolite of [(3)H]serotonin (presumably produced in the extracellular space of the retina). This uptake is blocked specifically by N-acetyl-serotonin. After an intraocular injection of forskolin into the rabbit eye the serotonin content of the tissue is elevated and this is reflected in the demonstration of serotonin immunoreactivity associated with a subpopulation of amacrine cells. These cells correspond in size and position to the "indoleamine-accumulating cells" of the retina. Animals (goldfish, frog, pigeon) dark-adapted over a certain period manifest an elevated level of serotonin associated with their retinas compared with that in light-adapted animals. However, immunofluorescence histochemistry used to localize serotonin showed that the same neurones in the different retinas contain the amine, although the "fluorescence" was elevated in the dark-adapted retinas. In the light of the present data it is clear that what constitutes a "serotonergic" neurone cannot be restricted to the immunohistochemical demonstration of the amine. The evidence is persuasive for "serotonergic neurones" having the property to take up serotonin specifically by a high affinity transport process. What warrants serious consideration is whether this property is a characteristic of "serotonergic neurones" rather than the endogenous localization of serotonin without pharmacological pretreatment.

Genetic reconstitution of the high-affinity L-arabinose transport system.

Expression plasmids containing various portions of araFGH operon sequences were assayed for their ability to facilitate the high-affinity L-arabinose transport process in a strain lacking the chromosomal copy of this operon. Accumulation studies demonstrated that the specific induction of all three operon coding sequences was necessary to restore high-affinity L-arabinose transport. Kinetic analysis of this genetically reconstituted transport system indicated that it functions with essentially wild-type parameters. Therefore, L-arabinose-binding protein-mediated transport appears to require only two inducible membrane-associated components (araG and araH) in addition to the binding protein (araF).

Relationship between low- and high-affinity glucose transport systems of Saccharomyces cerevisiae.

The high-affinity glucose transport process in Saccharomyces cerevisiae whole cells was regulated by catabolite repression and inactivation. The low-affinity process was constitutive, and its activity was inhibited in proportion to the extent of derepression of the high-affinity process. The latter finding suggests that there is some regulatory relationship between the two processes.

Effects of ovariectomy and estradiol benzoate on high affinity choline uptake, ACh synthesis, and release from rat cerebral cortical synaptosomes

Several presynaptic processes were studied in cerebral cortical synaptosomes prepared from intact adult female rats or from ovariectomized animals that received 3 subcutaneous injections of either estradiol benzoate (10 μg/kg) or vehicle. Injections were given 1/day, every other day, and animals were sacrificed 1 h after the last injection. High affinity choline uptake and coupled acetylcholine (ACh) synthesis were reduced by ovariectomy, and restored to control levels by the estradiol benzoate injections. In contrast, low affinity choline uptake and depolarization-induced [ 3H]ACh release were unaffected by either ovariectomy or estradiol benzoate injections. These results suggest that changes in estradiol levels may alter the high affinity transport process regulating ACh synthesis in this tissue.

Presence of multiple sodium-dependent phosphate transport processes in proximal brush-border membrane.

Renal brush-border membrane phosphate transport was studied in early and late segments of the pig proximal tubule. Vesicles were prepared from early proximal tubules (outer cortical tissue) and late proximal tubules (outer medullary tissue). Sodium-dependent phosphate uptake into brush-border membrane vesicles was determined using voltage clamp at 5-6 s, 21 degrees C. Sodium-dependent D-glucose uptake was determined to verify the cortical and medullary tissue cuts. At pH 8.0 (pHi = pHo), two sodium-dependent phosphate transport systems were evident in the early proximal tubule: a high-affinity system [Km, 0.06 +/- 0.01 mM; maximal transport activity (Vmax), 3.6 +/- 1.1 nmol X mg protein-1 X min-1] and a low-affinity system (Km, 4.11 +/- 0.02 mM; Vmax, 9.7 +/- 0.7 nmol X mg protein-1 X min-1). In the late proximal tubule at pH 8.0, only a single high-affinity transport process (Km, 0.19 +/- 0.7 mM; Vmax, 3.4 +/- 0.5 nmol X mg protein-1 X min-1) was evident. D-Glucose kinetics at pH 7.0 revealed both a high-affinity (Km, 0.55 +/- 0.09 mM) and a low-affinity (Km, 20.09 +/- 1.39 mM) system in the early proximal segment and a single high-affinity (Km, 1.27 +/- 0.36 mM) process in the late segment. These data suggest that two systems, distinct in their affinities and capacities, are involved in both D-glucose and phosphate transport across the brush-border membrane of the early proximal tubule, but that only a single high-affinity system is present in the late segment.

Characterization of [ 3H]hemicholinium-3 binding associated with neuronal choline uptake sites in rat brain membranes

Hemicholinium-3 (HCh-3) is a potent and specific inhibitor of the high-affinity choline transport process (HAChT) localized on cholinergic neurons. In this study, the specific binding of [ 3H]HCh-3 (120 Ci/mmol) was characterized in crude synaptic membranes prepared from rat brain. The binding of [ 3H]HCh-3 to forebrain membranes was saturable, reversible and specific with an apparent K d under optimal conditions of 35 nM and a B max of 56 fmol/mg protein. The potency of various HAChT inhibitors correlated with their apparent affinities for the specific [ 3H]HCh-3 binding site. The specific binding of [ 3H]HCh-3 exhibited an uneven regional distribution in the adult rat brain that corresponded to the activity of the HAChT in these regions. Transsection of the fornix, which causes a degeneration of the septal hippocampal cholinergic pathway, resulted in comparable reductions of the specific [ 3H]HCh-3 binding and the specific activity of choline acetyltransferase, a presynaptic marker for cholinergic terminals in the hippocampal formation; the lesion did not affect the specific activity of glutamic acid decar☐ylase, a presynaptic marker for GABAergic neurons within the hippocampus. Maximal binding occurred in the presence of 200 mM NaCl: potassium, lithium, rubidium and calcium substituted poorly for sodium; and bromide, fluoride, iodide, sulfate and phosphate were less effective anions than chloride. Increasing concentrations of NaCl increased the affinity of the site for [ 3H]HCh-3 with no significant effect on the maximal number of sites; the enhancement of affinity was due to a selective slowing of the rate of dissociation of the ligand from its binding site. These findings indicate that [ 3H]HCh-3 binds to the carrier site mediating the HAChT on cholinergic neurons; thus, this radioligand may be a useful probe for investigating this presynaptic component (HAChT) of cholinergic neurons.