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.     

Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice

Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.     

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.     

Estrogen Effects on High-Affinity Choline Uptake in Primary Cultures of Rat Basal Forebrain

Basal forebrain cholinergic neurons (BFCNs) degenerate in aging and Alzheimer’s disease. It has been proposed that estrogen can affect the survival and function of BFCNs. This study characterized primary rat BFCN cultures and investigated the effect of estrogen on high-affinity choline uptake (HACU). BFCNs were identified by immunoreactivity to the vesicular acetylcholine transporter (VAChT) and represented up to 5% of total cells. HACU was measured in living BFCN cultures and differentiated from low-affinity choline uptake by hemicholinium-3 (HC-3) inhibition. A HC-3 concentration curve showed that 0.3 μM HC-3, but not higher concentrations that inhibit LACU, could distinguish the two transport activities. 17-β-Estradiol treatment increased HACU in some culture preparations that contained non-neuronal cells. Elimination of dividing cells using antimitotic treatments resulted in a lack of estrogen effects on HACU. These results suggest that estrogen may have indirect effects on BFCNs that are mediated through non-neuronal cells.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.     

Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter

Regulation of intra- and extracellular ion activities (e.g. H(+), Cl(-), Na(+)) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na(+)/HCO(3)(-) cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO(3)(-) transporters are known: a neutral Na(+)/ HCO(3)(-) cotransporter, a K(+)/ HCO(3)(-) cotransporter, and a Na(+)-dependent Cl(-)-HCO(3)(-) exchanger. We report the cloning and characterization of a Na(+)-coupled Cl(-)-HCO(3)(-) exchanger and a physiologically unique bicarbonate transporter superfamily member. This Drosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughout Drosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl(-), Na(+), H(+), and HCO(3)(-) but does not require HCO(3)(-). Transport is blocked by the stilbene 4,4'-diisothiocyanodihydrostilbene- 2, 2'-disulfonates and may not be strictly electroneutral. Our functional data suggest this Na(+) driven anion exchanger (NDAE1) is responsible for the Na(+)-dependent Cl(-)-HCO(3)(-) exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva.     

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.     

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.     

Cortical choline transporter function measured in vivo using choline-sensitive microelectrodes: clearance of endogenous and exogenous choline and effects of removal of cholinergic terminals

The capacity of the high-affinity choline transporter (CHT) to import choline into presynaptic terminals is essential for acetylcholine synthesis. Ceramic-based microelectrodes, coated at recording sites with choline oxidase to detect extracellular choline concentration changes, were attached to multibarrel glass micropipettes and implanted into the rat frontoparietal cortex. Pressure ejections of hemicholinium-3 (HC-3), a selective CHT blocker, dose-dependently reduced the uptake rate of exogenous choline as well as that of choline generated in response to terminal depolarization. Following the removal of CHTs, choline signal recordings confirmed that the demonstration of potassium-induced choline signals and HC-3-induced decreases in choline clearance require the presence of cholinergic terminals. The results obtained from lesioned animals also confirmed the selectivity of the effects of HC-3 on choline clearance in intact animals. Residual cortical choline clearance correlated significantly with CHT-immunoreactivity in lesioned and intact animals. Finally, synaptosomal choline uptake assays were conducted under conditions reflecting in vivo basal extracellular choline concentrations. Results from these assays confirmed the capacity of CHTs measured in vivo and indicated that diffusion of substrate away from the electrode did not confound the in vivo findings. Collectively, these results indicate that increases in extracellular choline concentrations, irrespective of source, are rapidly cleared by CHTs.     

Effects of K+-Depolarization,Arachidonic Acid,Ethanol, and Aging on the High-Affinity Choline Transport in Rat Hippocampus

The Na⁺-dependent high-affinity choline uptake (HACU) transport and the [³H]hemicholinium-3 ([³H]HC-3) specific binding were measured on hippocampal synaptosomes of young (3–6 months) and old (22 months) Wistar rats. In vitro effects of 100–300 M arachidonic acid (AA) and of 5% ethanol were tested under basal as well as stimulated (55 mM KCl) conditions. The influence of AA (an irreversible decrease of HACU and a reversible increase of [³H]HC-3 binding) was more marked under stimulated rather than basal conditions in brain tissue of young rats. The increased K⁺-depolarization effect on HACU and the decreased influence of AA on [³H]HC-3 binding were estimated in brain tissue of old compared to young rats. Results suggest the involvement of different pools of the high-affinity choline carrier and marked changes due to aging in the regulation of the HACU transport.     

SLC5A9/SGLT4, a new Na+-dependent glucose transporter, is an essential transporter for mannose, 1,5-anhydro-D-glucitol, and fructose

We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) > D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).     

Age- and Sex-Dependent Effects of Ethanol on Hippocampal Hemicholinium-3 Sensitive Choline Carriers During Postnatal Development of Rats

Vulnerability of hippocampal hemicholinium-3 (HC-3)-sensitive carriers to ethanol was evaluated in vitro during rat postnatal development. The high-affinity uptake of [³H]choline (HACU) and the specific binding of [³H]HC-3 were measured on synaptosomes from 7-, 14-, and 60-day- and 3-month-old male and female Wistar rats. Marked increases of basal (between 7 and 60 days of age) and of stimulated HACU levels via K⁺-depolarization (between 14 days and 3 months) but only a mild elevation in [³H]HC-3 binding (between 7 days and 3 months) associated with alterations in the binding site number were found. On the mature tissue, ethanol at high concentrations (5%) moderately inhibited the choline transport under basal conditions but totally eliminated depolarization effects. However, both age- and sex-dependent alterations in basal HACU mediated by high or low pharmacologically relevant alcohol concentrations (50–100 mM) were observed in the immature tissue. Namely, the dose- and incubation time–dependent inhibition of HACU associated with changes in the transport velocity was found in postnatal male but not female tissue. [³H]HC-3 binding site was not markedly sensitive to ethanol actions. Anisotropy measurements in the region of the hydrophilic heads of phospholipid bilayers and in the membrane hydrocarbon core indicated penetration of 100 mM ethanol to immature female but not male tissue. Our results suggest the noncompetitive binding of alcohol to choline carriers from immature male tissue and correspond with data reporting significant sexual dimorphism of postnatal hippocampal neurons. The direct effects of ethanol on male choline carriers can contribute to the inhibition of acetylcholine synthesis and to sex-dependent neurotoxic effects of alcohol applied in vivo during early and late postnatal period.     

High-Affinity [3H]Choline Accumulation in Cultured Human Skin Fibroblasts

[3H]Choline can be transported across cell membranes by high-affinity (KT less than 5 microM) and low-affinity (KT much greater than 5 microM) systems. High-affinity choline accumulation (HACA) has been demonstrated in synaptosomes made from cholinergic brain regions such as the hippocampus and caudate-putamen. In cell culture, HACA has been demonstrated in glia and avian telencephalon, dissociated spinal cord, and muscle fibroblasts. We examined [3H]choline accumulation in a single normal human fibroblast line cultured from skin biopsy. [3H]Choline accumulation was temperature-dependent and linear with incubation time up to 6 min at 0.125 microM-choline. The apparent KT for [3H]choline was 5 microM, which is similar to that observed in avian fibroblasts. Isoosmotic replacement of Na+ with either Li+ (144 mM) or sucrose (288 mM) severely reduced [3H]choline accumulation (by 70-90%). Pre-incubation with ouabain (100 microM), sodium orthovanadate (100 microM), or 2,4-dinitrophenol (100 microM), or replacement of Ca2+ by Mg2+ had little or no effect on subsequent [3H]choline accumulation. [3H]Choline accumulation was inhibited by hemicholinium-3 (HC-3); after pre-incubation in HC-3 at 37 degrees C for 10 min, the IC50 (at 0.125 microM-choline) was 5.6 microM. The HC-3 sensitivity, Na+ dependence, and low KT suggest that human skin fibroblasts have a high-affinity transport system for choline.     

Identification of Sodium-Dependent, High-Affinity Choline Uptake Sites in Rat Brain with [3H]Hemicholinium-3

[3H]Hemicholinium-3 (HC-3) was used to label sodium-dependent, high-affinity choline uptake sites in regions of rat brain. Autoradiography revealed a high density of [3H]HC-3 binding sites in brain regions with a high density of cholinergic terminals, such as the interpeduncular nucleus, caudate-putamen, and olfactory tubercle. This distribution of [3H]HC-3 binding sites was in close agreement with the amounts of choline acetyltransferase in specific nuclei and subregions of rat brain. Destruction of presynaptic cholinergic projections in the cerebral cortex and the basal ganglia by injection of excitotoxins reduced [3H]HC-3 binding by 40-50%. These data indicate that sodium-dependent [3H]HC-3 binding sites are related to the choline transport system present in cholinergic neurons.     

Intra-Retrosplenial Cortical Grafts of Cholinergic Neurons: Functional Incorporation and Restoration of High Affinity Choline Uptake

Fetal septal neurons transplanted into the deafferented retrosplenial cortex (RSC) of rats have been shown to reinnervate the host brain and ameliorate spatial memory deficits. In the present study we examined the effects of implanting cholinergic neurons on high affinity choline uptake (HACU) in the denervated RSC and the correlational relationship between this cholinergic parameter and the level of behavioral recovery. Three groups of animals were used: 1) normal control rats (NC), 2) rats with lesions of the fornix and cingulate pathways (FX), and 3) lesioned rats with fetal septal grafts in the RSC (RSCsep-TPL). We found that intra-RSC septal grafts produced significant increases in HACU, and that recovery of HACU was significantly correlated with the improvements in the performance of spatial reference memory, spatial navigation, and spatial working memory tasks. We have also investigated the ability of the host brain to modulate the activity of the implanted neurons. In particular we evaluated the effect of the animals' performance in a 6-arm radial maze task on high affinity choline uptake (HACU). Animals in each of the NC, FX, and RSCsep-TPL groups were randomly assigned one of the following subgroups: 1) rats that performed the maze task before the determination of HACU (BEH), or 2) rats that did not perform the maze task before the determination of HACU (NON-BEH). Significant increases were observed in the NC and RSCsep-TPL groups, but not in the FX animals, indicating that fetal septal grafts in the RSC can become functionally incorporated with the host neural circuitry, and that the activity of the implanted cholinergic neurons can be modulated by the host brain.     

Role of Cl- in electrogenic Na+-coupled cotransporters GAT1 and SGLT1

We have investigated the functional role of Cl(-) in the human Na(+)/Cl(-)/gamma-aminobutyric acid (GABA) and Na(+)/glucose cotransporters (GAT1 and SGLT1, respectively) expressed in Xenopus laevis oocytes. Substrate-evoked steady-state inward currents were examined in the presence and absence of external Cl(-). Replacement of Cl(-) by gluconate or 2-(N-morpholino)ethanesulfonic acid decreased the apparent affinity of GAT1 and SGLT1 for Na(+) and the organic substrate. In the absence of substrate, GAT1 and SGLT1 exhibited charge movements that manifested as pre-steady-state current transients. Removal of Cl(-) shifted the voltage dependence of charge movements to more negative potentials, with apparent affinity constants (K(0.5)) for Cl(-) of 21 and 115 mm for SGLT1 and GAT1, respectively. The maximum charge moved and the apparent valence were not altered. GAT1 stoichiometry was determined by measuring GABA-evoked currents and the unidirectional influx of (36)Cl(-), (22)Na(+), or [(3)H]GABA. Uptake of each GABA molecule was accompanied by inward movement of 2 positive charges, which was entirely accounted for by the influx of Na(+) in the presence or absence of Cl(-). Thus, the GAT1 stoichiometry was 2Na(+):1GABA. However, Cl(-) was transported by GAT1 because the inward movement of 2 positive charges was accompanied by the influx of one Cl(-) ion, suggesting unidirectional influx of 2Na(+):1Cl(-):1GABA per transport cycle. Activation of forward Na(+)/Cl(-)/GABA transport evoked (36)Cl(-) efflux and was blocked by the inhibitor SKF 89976A. These data suggest a Cl(-)/Cl(-) exchange mechanism during the GAT1 transport cycle. In contrast, Cl(-) was not transported by SGLT1. Thus, in both GAT1 and SGLT1, Cl(-) modulates the kinetics of cotransport by altering Na(+) affinity, but does not contribute to net charge transported per transport cycle. We conclude that Cl(-) dependence per se is not a useful criterion to classify Na(+) cotransporters.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).