Par-4 inhibits choline uptake by interacting with CHT1 and reducing its incorporation on the plasma membrane

CHT1 is a Na(+)- and Cl(-)-dependent, hemicholinium-3 (HC-3)-sensitive, high affinity choline transporter. Par-4 (prostate apoptosis response-4) is a leucine zipper protein involved in neuronal degeneration and cholinergic signaling in Alzheimer's disease. We now report that Par-4 is a negative regulator of CHT1 choline uptake activity. Transfection of neural IMR-32 cells with human CHT1 conferred Na(+)-dependent, HC-3-sensitive choline uptake that was effectively inhibited by cotransfection of Par-4. Mapping studies indicated that the C-terminal half of Par-4 was physically involved in interacting with CHT1, and the absence of Par-4.CHT1 complex formation precluded the loss of CHT1-mediated choline uptake induced by Par-4, indicating that Par-4.CHT1 complex formation is essential. Kinetic and cell-surface biotinylation assays showed that Par-4 inhibited CHT1-mediated choline uptake by reducing CHT1 expression in the plasma membrane without significantly altering the affinity of CHT1 for choline or HC-3. These results suggest that Par-4 is directly involved in regulating choline uptake by interacting with CHT1 and by reducing its incorporation on the cell surface.     

High-Affinity Choline Transporter

The cholinergic neurons have long been a model for biochemical studies of neurotransmission. The components responsible for cholinergic neurotransmission, such as choline acetyltransferase, vesicular acetylcholine transporter, nicotinic and muscarinic acetylcholine receptors, and acetylcholine esterase, have long been defined as functional units and then identified as molecular entities. Another essential component in the cholinergic synapses is the one responsible for choline uptake from the synaptic cleft, which is thought to be the rate-limiting step in acetylcholine synthesis. A choline uptake system with a high affinity for choline has long been assumed to be present in cholinergic neurons. Very recently, the molecular entity for the high-affinity choline transporter was identified and is designated CHT1. CHT1 mediates Na⁺- and Cl^–-dependent choline uptake with high sensitivity to hemicholinium-3. CHT1 has been characterized both at the molecular and functional levels and was confirmed to be specifically expressed in cholinergic neurons.     

Molecular cloning and functional characterization of a neuronal choline transporter from Trichoplusia ni

A cDNA encoding a high-affinity Na(+)-dependent choline transporter (TrnCHT) was isolated from the CNS of the cabbage looper Trichoplusia ni using an RT-PCR-based approach. The deduced amino acid sequence of the CHT cDNA predicts a 594 amino acid protein of 64.74 kDa prior to glycosylation. TrnCHT has 80%, 79%, 76%, and 58% amino acid identity to putative CHTs from Anopheles gambiae, Drosophila melanogaster and Apis mellifera, and a cloned CHT from Limulus polyphemus, respectively. In situ hybridization of TrnCHT cRNA in whole-mount preparations of caterpillar CNS revealed that TrnCHT mRNA is expressed by hundreds of presumably cholinergic neurons present in both the brain and cortex of all segmental ganglia. Na(+)-dependent [(3)H]-choline uptake was induced in Sf9 cells in vitro following infection with a TrnCHT-expressing recombinant baculovirus. Virally induced [(3)H]-choline uptake was found to approximately equal the endogenous rate of choline uptake in insect cells, seen either after infection with a control virus or in TrnCHT-infected cells exposed to [(3)H]-choline in the absence of Na(+). The Na(+)-dependent component of [(3)H]-choline uptake by TrnCHT-infected cells was saturable with a K(m) for choline transport of 8.4 microM. Several compounds reported to be potent blockers of [(3)H]-choline uptake by cloned vertebrate choline transporters proved to be relatively weak inhibitors of choline uptake by Sf9 cells expressing TrnCHT. Hemicholinium-3 (K(i)=4.1 microM) and two oxoquinuclidium analogues of choline, quireston-A (K(i) approximately 10 microM) and quireston (K(i) approximately 100 microM) inhibited 50% of control uptake only at micromolar concentrations. The endogenous low-affinity Na(+)-independent uptake of [(3)H]-choline was also inhibited by high micromolar concentrations of hemicholinium-3.     

A conserved asparagine residue in transmembrane segment 1 (TM1) of serotonin transporter dictates chloride-coupled neurotransmitter transport

Na(+)- and Cl(-)-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl(-) coordination of human serotonin transport have been identified, the role of Cl(-) in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl(-) binding to concentrative neurotransmitter uptake.     

Renal transport of taurine in luminal membrane vesicles from rabbit proximal tubule

The uptake of taurine by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule was examined. In pars convoluta, the transport of taurine was characterized by two Na(+)-dependent (Km1 = 0.086 mM, Km2 = 5.41 mM) systems, and one Na(+)-independent (Km = 2.87 mM) system, which in the presence of an inwardly directed H(+)-gradient was able to drive the transport of taurine into these vesicles. By contrast, in luminal membrane vesicles from pars recta, the transport of taurine occurred via a dual transport system (Km1 = 0.012 mM, Km2 = 5.62 mM), which was strictly dependent on Na+. At acidic pH with or without a H(+)-gradient, the Na(+)-dependent flux of taurine was drastically reduced. In both kind of vesicles, competition experiments only showed inhibition of the Na(+)-dependent high-affinity taurine transporter in the presence of beta-alanine, whereas there was no significant inhibition with alpha-amino acids, indicating a beta-amino acid specific transport system. Addition of beta-alanine, L-alanine, L-proline and glycine, but not L-serine reduced the H(+)-dependent uptake of taurine to approx. 50%. Moreover, only the Na(+)-dependent high-affinity transport systems in both segments specifically required Cl-. Investigation of the stoichiometry indicated 1.8 Na+: 1 Cl-: 1 taurine (high affinity), 1 Na+: 1 taurine (low affinity) and 1 H+: 1 taurine in pars convoluta. In pars recta, the data showed 1.8 Na+: 1 Cl-: 1 taurine (high affinity) and 1 Na+: 1 taurine (low affinity).     

Expression of rat liver glutamine transporters in Xenopus laevis oocytes.

As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.     

Carnitine/xenobiotics transporters in the human mammary gland epithelia, MCF12A

The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of human (h) novel organic cation transporters (OCTN) 1 and hOCTN2 (a Na+-dependent carnitine carrier with Na+-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC OCTs such as hOCT1 and extraneuronal monoamine transporter (EMT)/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of ATB0+ (an amino acid transporter with a Na+/Cl(-)-dependent carnitine transport activity), and Fly-like putative transporter 2/OCT6 (a splice variant of carnitine transporter 2: a testis-specific Na+-dependent carnitine transporter). TEA uptake was pH dependent. Carnitine uptake was dependent on Na+, and partly on Cl-, compatible with hOCTN2 and ATB0+ function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by K(m) of 5.1 microM for carnitine and 1.6 mM for TEA, apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake but required supratherapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotic challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/nutrient transporters in human mammary gland drug transfer.     

A Na+-phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter

The SLC17 anion transporter family comprises nine members that transport various organic anions in membrane potential (Δψ)- and Cl(-)-dependent manners. Although the transport substrates and physiological relevance of the majority of the members have already been determined, little is known about SLC17A4 proteins known to be Na(+)-phosphate cotransporter homologue (NPT homologue). In the present study, we investigated the expression and transport properties of human SLC17A4 protein. Using specific antibodies, we found that a human NPT homologue is specifically expressed and present in the intestinal brush border membrane. Proteoliposomes containing the purified protein took up radiolabeled p-aminohippuric acid (PAH) in a Cl(-)-dependent manner at the expense of an electrochemical gradient of protons, especially Δψ, across the membrane. The Δψ- and Cl(-)-dependent PAH uptake was inhibited by diisothiocyanostilbene-2,2'-disulfonic acid and Evans blue, common inhibitors of SLC17 family members. cis-Inhibition studies revealed that various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs, pravastatin, and urate inhibited the PAH uptake. Proteoliposomes took up radiolabeled urate, with the uptake having properties similar to those of PAH uptake. These results strongly suggested that the human NPT homologue acts as a polyspecific organic anion exporter in the intestines. Since SLC17A1 protein (NPT1) and SLC17A3 protein (NPT4) are responsible for renal urate extrusion, our results reveal the possible involvement of a NPT homologue in urate extrusion from the intestinal duct.     

Functional expression of choline transporter-like protein 1 (CTL1) in human neuroblastoma cells and its link to acetylcholine synthesis

We examined the molecular and functional characterization of choline uptake into human neuroblastoma cell lines (SH-SY5Y: non-cholinergic and LA-N-2: cholinergic neuroblastoma), and the association between choline transport and acetylcholine (ACh) synthesis in these cells. Choline uptake was saturable and mediated by a single transport system. Removal of Na(+) from the uptake buffer strongly enhanced choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium. The increase in choline uptake under Na(+)-free conditions was inhibited by a Na(+)/H(+) exchanger (NHE) inhibitor. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), NHE1 and NHE5 mRNA are mainly expressed. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. ChAT mRNA was expressed at a much higher level in LA-N-2 cells than in SH-SY5Y cells. The conversion of choline to ACh was confirmed in both cells, and was enhanced in Na(+)-free conditions. These findings suggest that CTL1 is functionally expressed in both SH-SY5Y and LA-N-2 cells and is responsible for choline uptake that relies on a directed H(+) gradient as a driving force, and this transport functions in co-operation with NHE1 and NHE5. Furthermore, choline uptake through CTL1 is associated with ACh synthesis in cholinergic neuroblastoma cells.     

Differential effect of pH on sodium binding by the various GABA transporters expressed in Xenopus oocytes

Mouse GABA transporters belong to the family of Na(+)- and Cl(-)-dependent neurotransmitter transporters. The four GABA transporters exhibit unique presteady-state currents when expressed in Xenopus oocytes. The properties of the presteady-state currents correspond to their different affinities to Na(+). In the presence of 20 microM GABA and at pH 7.5, the half-maximal uptake activity was 47, 120, 25 and 35 mM Na(+) for GAT1, GAT2, GAT3 and GAT4, respectively. The appearance of presteady-state currents at positive or negative imposed potentials was in correlation with the affinity to Na(+). Changing the external pH differentially affected the GABA uptake and the presteady-state activities of the various GABA transporters. It is suggested that protons compete with Na(+) on its binding site; however, the proton binding is not productive and is unable to drive GABA uptake.     

胆碱转运体与阿尔茨海默病

阿尔茨海默病主要病理学特征是在脑中形成大量的老年斑和神经元纤维缠结以及出现弥漫性脑萎缩.胆碱能系统的失调与阿尔茨海默病的发生机制关系密切.具体表现为基底前脑的胆碱能系统紊乱,胆碱乙酰化酶、乙酰胆碱含量显著减少,以及大量胆碱能神经元退化.胆碱转运体是胆碱能系统中用于转运胆碱进入细胞的关键蛋白体,有三种类型:高亲和力胆碱转运体、胆碱转运体类蛋白及非特异性有机阳离子转运体.近年,很多研究表明胆碱转运体的异常与一系列神经退行性紊乱有关.本文简要综述胆碱能系统中胆碱转运体的生理作用及其在阿尔茨海默病中异常代谢和可能机制的研究进展,以期为防治阿尔茨海默病提供进一步的理论和实验依据.     

Existence of an endogenous glutamate and aspartate transporter in Chinese hamster ovary cells

Chinese hamster ovary cells show endogenous high-affinity Na^＋ -dependent glutamate transport activity. This transport activity is kinetically similar to a glutamate transporter family strategically expressed in the central nervous system and is pharmacologically unlike glutamate transporter- 1 or excitatory amino acid carrier 1. The cDNA of a glutamate/aspartate transporter （GLAST）-like transporter was obtained and analyzed. The deduced amino acid sequence showed high similarity to human, mouse, and rat GLAST. We concluded that a GLAST-like glutamate transporter exists in Chinese hamster ovary cells that might confer the endogenous high-affinity Na^＋ -dependent glutamate transport activity evident in these cells.     

A high-affinity octopamine transporter cloned from the central nervous system of cabbage looper Trichoplusia ni.

A cDNA encoding a high-affinity Na(+)/anion(-)-dependent octopamine transporter (OAT) was isolated via an RT-PCR-based approach from caterpillars of the cabbage looper, Trichoplusia ni. The deduced amino acid sequence of the OAT cDNA predicts a 670 amino acid protein bearing strong homology to previously cloned monoamine transporters. The expression pattern of OAT mRNA in the central nervous system revealed by in situ hybridization closely resembles that of OA-ergic neurons identified by the presence of mRNA for tyramine beta-hydroxylase, a marker enzyme for OA-ergic neurons in invertebrates. In vitro, insect cells infected with OAT-expressing baculovirus accumulated both (3)H-OA and (3)H-dopamine with saturation kinetics typical of carrier-mediated processes. (3)H-dopamine uptake by OAT was most inhibited by tyramine, OA, dopamine and the tricyclic antidepressants desipramine and imipramine. Substitution studies for Na(+) and Cl(-) indicate that OAT has a strong requirement for Na(+) and a less stringent requirement for Cl(-). The pharmacological profile of OAT is distinct from those of other cloned monoamine transporters and makes OAT a potential target for neuro-active pest control agents.     

Molecular and functional characterization of an Na+-independent choline transporter in rat astrocytes

In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (Km) of 35.7 +/- 4.1 microm and a maximal velocity (Vmax) of 49.1 +/- 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17beta-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.     

Expression of Na(+) / D-glucose cotransport in Xenopus laevis oocytes by injection of poly(A)(+) RNA isolated from lobster (Homarus americanus) hepatopancreas

Xenopus laevis oocytes were used for expression and characterization of lobster (Homarus americanus) hepatopancreas Na(+)-dependent D-glucose transport activity. Poly(A)(+) RNA from the whole hepatopancreatic tissue was injected and transport activity was assayed by alpha-D-[2-(3)H] glucose. Injection of lobster hepatopancreatic poly(A)(+) RNA resulted in a dose (1-20 ng) and time (1-5 days) dependent increase of Na(+)-dependent D-glucose uptake. Kinetics of Na(+)-dependent glucose transport was a hyperbolic function (K(m)=0.47+/-0.04 mM) of external D-glucose concentration and a sigmoidal function (K(Na)=68.32+/-1.57 mM; Hill coefficient=2.22+/-0.09) of external Na(+) concentration. In addition, Na(+)-dependent D-glucose uptake was significantly inhibited by both (0.1-0.5 mM) phloridzin and (0.1-0.5 mM) methyl-alpha-D-glucopyranoside. After size fractionation through a sucrose density gradient, poly(A)(+) RNA fractions with an average length of 2-4 kb induced a twofold increase in Na(+)-dependent phloridzin-inhibited D-glucose uptake as compared to total poly(A)(+) RNA-induced uptake. The results of this study provide the functional basis to screen lobster hepatopancreatic cDNA libraries for clones encoding putative and still not known crustacean SGLT-type Na(+)/glucose co-transporter(s).     

Effect of sodium lithium and proton concentrations on the electrophysiological properties of the four mouse GABA transporters expressed in Xenopus oocytes

Mouse GABA transporters belong to the family of Na(+) and Cl(-) dependent neurotransmitter transporter. GABA transport, by these family members, was shown to be electrogenic and driven by sodium ions. It was demonstrated that, as in several other transporters, sodium binding and release by GAT1, GAT3 and BGT-1, the canine homolog of GAT2, resulted in the appearance of presteady-state currents. In this work we show that each of the four GABA transporters exhibit unique presteady-state currents when expressed in Xenopus oocytes. The properties of the presteady-state currents correspond to the transporters affinities to Na(+). At 100 mM GAT1 exhibited symmetric presteady-state currents at all imposed potentials, whereas GAT2 exhibited asymmetric presteady-state currents exclusively at negative imposed potentials, GAT3 or GAT4 exhibited presteady-state currents predominantly at positive imposed potentials. GABA uptake by GAT2 and GAT4 was much more sensitive to external pH than GAT1 and GAT3. Reducing the external Na(+) concentration rendered the GABA uptake activity by GAT1 and GAT3 to be sensitive to pH. Lowering the external pH reduced the Na(+) affinity of GAT1. Substitution of the external Na(+) to Li(+) resulted in the appearance of leak currents exclusively at negative potentials in Xenopus oocyte expressing GAT1 and GAT3. Low Na(+) concentrations inhibited the leak currents of GAT1 but Na(+) had little effect on the leak currents of GAT3. Washing of occluded Na(+) in GAT1 enhanced the leak currents. Similarly addition of GABA in the presence of 80 mM Li(+), that presumably accelerated the release of the bound Na(+), also induced the leak currents. Conversely, addition of GABA to GAT3 expressing oocytes, in the presence of 80 mM Li(+), inhibited the leak currents.     

Functional regulation of Na+-dependent neutral amino acid transporter ASCT2 by S-nitrosothiols and nitric oxide in Caco-2 cells

We describe the regulation mechanisms of the Na(+)-dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco-2. Exposure of Caco-2 cells to S-nitrosothiol, such as S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione, and the NO-donor, NOC12, concentration- and time-dependently increased Na(+)-dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (V(max)) of Na(+)-dependent alanine uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na(+)-dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) and N-(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT-PCR revealed that Caco-2 cells expressed the Na(+)-dependent neutral amino acid transporter ASCT2, but not the other Na(+)-dependent neutral amino acid transporters ATB(0,+) and B(0)AT1. These results suggested that functional up-regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis.     

Stereospecificity of High-and Low-Affinity Transport of Choline Analogues into Rat Cortical Synaptosomes

The present experiments used methylcholines to examine the stereoselectivity of choline transport into rat synaptosomes. R(+)-alpha-methylcholine and S(+)-beta-methylcholine were significantly better inhibitors of the high-affinity choline transport system than were their enantiomers. Although both enantiomers of alpha- and of beta-methylcholine inhibited [3H]choline transport, only R(+)-alpha-methylcholine and S(+)-beta-methylcholine could be transported by the high-affinity choline uptake mechanism. Therefore, we conclude that the chiral requirements for recognition of and for transport by the high-affinity transporter are clearly different. In addition to high-affinity choline transport, Na(+)-independent low-affinity transport was measured. This process transported R(+)-alpha-methylcholine, but not S(-)-alpha-methylcholine; however, it showed no stereoselectivity for the enantiomers of beta-methylcholine. Thus, high- and low-affinity choline transport mechanisms exhibit distinct differences in their substrate selectivities. We suggest that the stereoselective properties of choline transport might present a unique opportunity to study choline uptake and metabolism.