Vesicular Localization and Activity-Dependent Trafficking of Presynaptic Choline Transporters

Abstract

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.

Choline Transport and de novo Choline Synthesis Support Acetylcholine Biosynthesis in Caenorhabditis elegans Cholinergic Neurons

“…Similar results were reported recently by Matthies et al (2006). In mice, a significant fraction of CHT protein appears to be associated with cholinergic synaptic vesicles (Ferguson et al 2003). This observation has led to an appealing model of CHT trafficking and function, whereby the process of releasing vesicular ACh simultaneously delivers CHT to the plasma membrane (Ferguson et al 2003;reviewed in Ferguson and Blakely 2004).…”

“…Similarly, CHT proteins in rats, mice, and humans are expressed in most cholinergic neurons (Misawa et al 2001;Ferguson et al 2003), consistent with their presumed primary role in providing choline for ACh synthesis. In the present study, we also noted expression of CHO-1 in several (apparently) noncholinergic neurons, as well as in the intestine.…”

“…However, as noted earlier, researchers have also reported that a small, but significant fraction ($20%) of ACh synthesis is independent of HC-3-sensitive choline uptake (Birks and Macintosh 1957;Guyenet et al 1973). Other researchers have demonstrated that neuroendocrine cells are capable of synthesizing and releasing ACh in the absence of detectable CHTexpression (Bauerfeind et al 1993;Ferguson et al 2003). These observations are generally attributed to the activity of low-affinity choline transporters .…”

“…In mice, a significant fraction of CHT protein appears to be associated with cholinergic synaptic vesicles (Ferguson et al 2003). This observation has led to an appealing model of CHT trafficking and function, whereby the process of releasing vesicular ACh simultaneously delivers CHT to the plasma membrane (Ferguson et al 2003;reviewed in Ferguson and Blakely 2004). We found that the distribution of CHO-1 only partially overlaps with that of VAChT (Figure 3), suggesting that a significant fraction of CHO-1 is not present on cholinergic synaptic vesicles.…”

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1

“…To understand the extent of this influence, it is essential to understand the mechanisms underlying subcellular transporter trafficking. We and others have previously isolated transporter-containing synaptic-like vesicles that are potential mediators of transporter trafficking in axon terminals (10), and we have identified many signaling molecules that regulate subcellular transporter redistribution (12,14,15,22,24,28). In this report, by using a modified biotinylation assay to study GAT1 trafficking, (i) we defined an acutely recycling pool of GAT1 in cortical neurons that, in the basal state, comprises approximately one-third of total cellular GAT1; (ii) we measured the exocytosis rate (r exo ϭ 0.7 min Ϫ1 ) and the endocytosis rate (r endo ϭ 1.1 min Ϫ1 ) of acutely recycling GAT1 in the basal state; and (iii) we demonstrated that distinct transporter redistribution signals exert their effects by differentially regulating the recycling pool size or selectively uncoupling rates of exocytosis and endocytosis.…”