Aberrant trafficking of the high-affinity choline transporter in AP-3-deficient mice.
The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.
Department of Pharmacology, Kyoritsu University of Pharmacy, Tokyo 105-8512, Japan. misawa-hd@kyoritsu-ph.ac.jp
Hidemi
H
Misawa
Hirofumi
H
Fujigaya
Takashi
T
Nishimura
Yasuhiro
Y
Moriwaki
Takashi
T
Okuda
Koichiro
K
Kawashima
Kazuko
K
Nakata
Alicia M
AM
Ruggiero
Randy D
RD
Blakely
Fubito
F
Nakatsu
Hiroshi
H
Ohno
D000109:Acetylcholine / Q000096:biosynthesis / Q000378:metabolism*; D033963:Adaptor Protein Complex 3 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002794:Choline / Q000378:metabolism; D002799:Cholinergic Fibers / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism; D016716:PC12 Cells; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D050794:STAT1 Transcription Factor / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism*; D014162:Transfection
D033963:Adaptor Protein Complex 3; C488255:CHT1 protein, mouse; D026901:Membrane Transport Proteins; D050794:STAT1 Transcription Factor; D002794:Choline; D000109:Acetylcholine