Identification, subcellular localization and targeted disruption of the presynaptic, hemicholinium-3-sensitive choline transporter

Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3 (HC-3) sensitive choline transporter (CHT). In the studies comprising this thesis, I describe the molecular cloning and characterization of human and murine CHT cDNAs. Following the development of CHT-specific antibodies, we observed an exclusive localization of CHT to cholinergic neurons and demonstrated that the majority of CHTs in the rat and mouse brain reside on small vesicles within cholinergic presynaptic terminals. Furthermore, gradient fractionation and immunoisolation of presynaptic vesicles using multiple CHT and control antibodies revealed that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT), contain ACh and represent a distinct subset of cholinergic synaptic vesicles. Additionally, we found that depolarization of synaptosomes evoked a Ca ++ dependent, botulinum neurotoxin C (BoNt/C) sensitive increase in the Vmax for HC-3 sensitive choline uptake accompanied by an increase in the density of CHTs in synaptic plasma membranes. Targeted disruption of the CHT gene in mice abolished HC-3-sensitive choline uptake, resulting in impaired ACh release at neuromuscular junctions (NMJs) and neonatal lethality in CHT-/- mice. Moreover, CHT+/- mice exhibited normal gross morphology, behavior and fertility despite a partial loss of CHT protein abundance. [3H]-Choline uptake and [3H]-HC-3 binding were unaltered, relative to wildtype animals, in whole brain synaptosome and membrane preparations from CHT+/- mice. These studies reveal the presence of post-translational regulatory mechanisms that can monitor the functional CHT capacity in neurons and support normal choline uptake in the context of significant losses of CHT protein. My studies suggest that the mechanisms supporting regulation of presynaptic CHT function could be targets for novel therapeutics that may offset functional deficits in disorders bearing diminished cholinergic tone, including myasthenias and dementias.