Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.

Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration. Key words: serotonin; norepinephrine; dopamine; transporter; protein phosphatase 2A; protein kinase C; phosphorylationThe biogenic amine neurotransmitters dopamine (DA), norepinephrine (NE), and serotonin (5HT) are important modulators of multiple motor, cognitive, and autonomic processes in the brain and periphery (Feldman et al., 1997;Deutch and Roth, 1999). Active termination of aminergic signaling is mediated by plasma membrane transport proteins of the Na ϩ /Cl Ϫ -dependent cotransporter gene family (Povlock and Amara, 1997;Nelson, 1998). Pharmacological blockade of DA transporter (DAT), NE transporter (NET), and serotonin transporter (SERT) proteins by psychostimulant (e.g., cocaine, amphetamine, methylphenidate) and antidepressant uptake inhibitors elevates extracellular neurotransmitter levels with subsequent augmentation of receptor stimulation (Barker and Blakely, 1995). Genetic ablation of DAT (Giros et al., 1996), NET (Xu et al., 2000), and SERT (Bengel et al., 1998) proteins reveals a profound contribution of appropriate transporter expression to presynaptic transmitter homeostasis and receptor stimulation. Recently, regulatory and coding polymorphisms have been identified in human SERT and NET genes and linked to affective disorders and autonomic disturbances, respectively (Lesch...

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