Vesicular localization and activity-dependent trafficking of presynaptic choline transporters.
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.
Neuroscience Graduate Program, Center for Molecular Neuroscience, Vanderbilt University, Nashville, Tennessee 37232, USA.
Shawn M
SM
Ferguson
Valentina
V
Savchenko
Subbu
S
Apparsundaram
Melissa
M
Zwick
Jane
J
Wright
Craig J
CJ
Heilman
Hong
H
Yi
Allan I
AI
Levey
Randy D
RD
Blakely
D000818:Animals; D000918:Antibody Specificity; D015415:Biomarkers / Q000032:analysis; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002799:Cholinergic Fibers / Q000737:chemistry / Q000378:metabolism; D007163:Immunosorbent Techniques; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D016716:PC12 Cells; D017729:Presynaptic Terminals / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D013347:Subcellular Fractions / Q000737:chemistry; D013572:Synaptic Vesicles / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D015415:Biomarkers; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C493460:Slc18a3 protein, mouse; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; C065637:choline transporter