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Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling.

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.

The involvement of protein kinase C (PKC) in the regulation of [3H]choline cotransport was studied in Limulus brain hemi-slice preparations. The PKC activators, phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu), significantly decreased [3H]choline cotransport. Conversely, the PKC inhibitors, staurosporine (STAURO) and polymyxin B (PMB), each increased [3H]choline cotransport. These PKC inhibitors prevented the phorbol ester-induced reduction of transport. Both the PMA induced decrease and the STAURO induced increase in [3H]choline cotransport were paralleled by respective and comparable changes in [3H]hemicholinium-3 (HC-3) specific binding. Pre-exposure of brain hemi-slices to elevated potassium chloride (120 mM KCl) resulted in a doubling of [3H]choline cotransport and [3H]HC-3 binding. The enhancement of [3H]choline cotransport by STAURO and antecedent 120 mM KCl treatment were additive. PMA did not significantly alter elevated potassium stimulated transport. Moreover, arachidonyltrifluoromethyl ketone (AACOCF3) and quinacrine (QUIN), both phospholipase A2 (PLA2) inhibitors, markedly decreased enhanced [3H]choline transport and [3H]HC-3 binding induced by antecedent exposure to depolarizing concentrations of potassium. These results suggest that PKC and PLA2 are involved in the regulation of [3H]choline cotransport but at different regulatory sites.

The cholinergic neurotoxin AF64A (ethylcholine aziridinium) has been used to selectively destroy the cholinergic system. Due to its structural similarity to choline, this compound may be selectively taken up by the cholinergic terminal via the high-affinity choline transport (HAChT) system to produce persistent and selective cholinergic deficits. The mechanism by which it exerts its cholinotoxicity remains to be elucidated. We have examined the effects of AF64A in the human neuroblastoma cell line, LA-N-2, which has an intact sodium-coupled choline uptake system, and is capable of synthesizing acetylcholine (ACh). AF64A (25, 50 and 100 μM) produced dose-dependent increases in cell kill as measured by colony formation assay. The addition of increasing concentrations (10 −5 , 10 −4 and 10 −3 M) of choline and hemicholinium-3 (HC-3) protected the cells from the cytotoxic effects of AF64A. At the same doses, AF64A also decreased choline acetyltransferase (ChAT) activity. In the presence of the highest concentration of choline or HC-3 (10 −3 M) which produced complete protection against AF64A's cytotoxicity in the colony formation assay, ChAT activity was restored to control values. These results demonstrate that agents that utilize (i.e., choline) or inhibit (i.e., HC-3) the choline uptake system prevented AF64A-induced cytotoxicity and decreases in ChAT activity, in a manner similar to that which has been observed in chick and rat primary cholinergic cultures in vitro. The LA-N-2 neuroblastoma cell line thus serves well as an in vitro model of the cholinergic neuron and provides a useful system to study the mode of cholinotoxicity induced by AF64A.

The sodium-dependent, high affinity choline transporter - choline cotransporter - (ChCoT, aka: cho-1, CHT1, CHT) undergoes constitutive and regulated trafficking between the plasma membrane and cytoplasmic compartments. The pathways and regulatory mechanisms of this trafficking are not well understood. We report herein studies involving selective endosomal ablation to further our understanding of the trafficking of the ChCoT. Selective ablation of early sorting and recycling endosomes resulted in a decrease of approximately 75% of [3H]choline uptake and approximately 70% of [3H]hemicholinium-3 binding. Western blot analysis showed that ablation produced a similar decrease in ChCoTs in the plasma membrane subcellular fraction. The time frame for this loss was approximately 2 h which has been shown to be the constitutive cycling time for ChCoTs in this tissue. Ablation appears to be dependent on the intracellular cycling of transferrin-conjugated horseradish peroxidase and the selective deposition of transferrin-conjugated horseradish peroxidase in early endosomes, both sorting and recycling. Ablated brain slices retained their capacity to recruit via regulated trafficking ChCoTs to the plasma membrane. This recruitment of ChCoTs suggests that the recruitable compartment is distinct from the early endosomes. It will be necessary to do further studies to identify the novel sequestration compartment supportive of the ChCoT regulated trafficking.

1. The distribution of several cholinergic components generally accepted as being associated with cholinergic transmission was studied in Limulus nerve and muscle tissue. 2. Acetylcholine (ACh) and choline were distributed among all the tissues assayed, with abdominal ganglia showing the highest concentration of both. 3. Corpora pedunculata showed the greatest enrichment of choline acetyltransferase (ChAT) and the brain nerve roots had the highest levels of acetylcholinesterase (AChE) activity. No direct correlation was found between levels of ChAT and the amount of measurable ACh in a tissue. 4. Tissue/medium (T/M) ratios indicative of high affinity [ 3 H]choline uptake were selectively distributed, ranging from 13.22 to 4.62 in corpora pedunculata, circumoesophageal ring, abdominal ganglia and cardiac ganglion. All these tissues, with the exception of cardiac ganglion, released [ 3 H]ACh in a Ca 2+ dependent fashion in response to treatment with elevated potassium (90 mM). 5. Cardiac ganglion was found not to synthesize [ 3 H]ACh and had no detectable levels of ChAT activity. 6. These results suggest that high affinity [ 3 H]choline uptake is not exclusively associated with the synthesis of [ 3 H]ACh and hence is not a unique property of cholinergic neurons and/or terminals.

The intent of this study was to determine whether the drug 2-(4-phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine-induced increase in acetylcholine (ACh) synthesis by reducing the veratridine-induced activation of a detergent-soluble choline-O-acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle-bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by l-AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of l-AH 5183 (75 nM), the depolarization-induced activation of a detergent-soluble ChAT fraction associated with a vesicle-bound store of ACh was blocked. Conversely, the veratridine-induced activation of a water-soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle-bound store with newly synthesized ACh following veratridine-induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergentsoluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183-sensitive regulatory protein.

1. The transport of [3H]choline by the corpora pedunculata of the circumoesophageal ring gland (brain) of Limulus polyphemus was studied. Corpora pedunculata slices were incubated individually in Chao's solution containing 0.01 μM [3H]choline at room temperature (25 ± 2°C) and readily accumulated the radiolabel from the extracellular environment. 2. The corpora pedunculata uptake of [3H]choline was linear over 60 min. 3. The kinetic analysis indicated the existence of dual uptake systems for choline within the corpora pedunculata, a high affinity choline uptake process (Km = 0.54 μM and Vmax = 0.037 pmoles/mg/min) and a low affinity process (Km = 137 μM and Vmax = 6.3 pmoles/mg/min). 4. The high affinity choline transport system was dependent on sodium ions and was inhibited by micromolar concentrations of hemicholinium-3. 5. The pre-exposure of the corpora pedunculata to Chao's solution containing 90 mM potassium for 15 min resulted in a 24% increase in the velocity of the high affinity choline uptake process (Vmax = 0.046 pmoles/mg/min). 6. The 90 mM potassium Chao's pretreatment stimulated a substantial increase in the synthesis of [3H]acetyleholine by the corpora pedunculata. 7. The results suggest that the high affinity choline uptake process within the Limulus corpora pedunculata is associated with the synthesis of the transmitter acetylcholine, presumably within cholinergic terminals in this tissue.

1. The Limulus cardiac ganglion high affinity choline uptake system (HAChUS) was inhibited 40, 51 and 64% following pre-exposure to 10, 100 and 500 μM vinblastine, respectively. 2. In contrast, high affinity uptake of choline in the Limulus corpora pedunculata and abdominal ganglia, tissues in which a cholinergic function has been described, were unaffected. 3. In pulse-chase experiments, the cardiac ganglion was incubated in 0.1 μM [ 3 H]choline for 60 min and then switched to an incubation medium containing 1 mM unlabelled choline for varying periods of time. 4. Under these conditions, a 3-fold increase of radiolabel above basal level was measured in the pellet fraction within 2 hr of post-labelling incubation. 5. Prior exposure of the ganglion to 500 μM vinblastine completely eliminated this increase of radioactivity in the pellet fraction. 6. Treatment of the radiolabelled pellet fraction with phospholipase C resulted in the solubilization of 72% of the radiolabel. 7. Ten (10) μM 5-hydroxytryptamine (5-HT), a concentration previously shown to inhibit spontaneous electrical activity within the cardiac ganglion, resulted in a 40% decrease in high affinity choline uptake in this tissue selectively. 8. These results are consistent with the view that a probable role of the Limulus cardiac ganglion HAChUS is the supply of choline subserving the synthesis of membrane phospholipid. 9. It is further speculated that this membrane phospholipid synthesis may be associated with synaptic vesicle turnover.

1. 1. The cardiac ganglion of the horseshoe crab, Limulus polyphemus , was incubated in Chao's solution containing 0.01 μM [ 3 H]choline at room temperature (25 ± 2°C) and the ganglion readily accumulated the radiolabel. 2. 2. The ganglion uptake of [ 3 H]choline was linear over 60 min. 3. 3. Kinetic analysis revealed dual choline uptake systems within the cardiac ganglion, a high affinity uptake system ( K m = 2.2 μ M, V max = 0.16pmoles/mg/min) and a low affinity system ( K m = 92.3 μ M, V max = 3.08 prnoles/mg/min). 4. 4. The high affinity uptake system was sodium-dependent and inhibited by micromolar concentrations of hemicholinium-3. 5. 5. A 15 min pre-exposure of the ganglion to Chao's solution containing 90 mM potassium stimulated a significant increase in choline uptake. 6. 6. There was no detectable synthesis of [ 3 H]acetylcholine from the [ 3 H]choline taken up by the cardiac ganglion. The major portion of the extraetable label appeared in a fraction which co-electrophoresed with phosphorylcholine. 7. 7. These results suggest that the sodium-dependent high affinity [ 3 H]choline uptake system of the cardiac ganglion subserves a specific requirement for choline which is unrelated to a cholinergic function.

1. Comparative studies of [ 3 H]choline accumulation were done in the Limulus corpora pedunculata, abdominal ganglia and cardiac ganglion. Dual uptake processes for choline were found in all three tissues. 2. In acute experiments, the corpora pedunculata high affinity choline uptake system showed exclusive sensitivity to ouabain. Prolonged exposure to ouabain revealed that the HAChUS of all three tissues were significantly inhibited. 3. The metabolism of [ 3 H]choline transported via the high affinity process in the three tissues was studied. [ 3 H]Acetylcholine was a major product of the [ 3 H]choline taken up by the corpora pedunculata and the abdominal ganglia. 4. Phosphorylcholine was the major product seen in cardiac ganglion extracts and occurred in significant proportions in abdominal ganglia extracts. 5. [ 3 H]Acetylcholine was not detected in cardiac ganglion extracts. 6. Treatment with either lithium chloride or hemicholinium-3 markedly inhibited high affinity uptake of [ 3 H]choline in all three tissues.