The acetylcholine storage system and drug inhibition in cholinergic synaptic vesicles

Elucidating fundamental principles of nervous system operations is the foundation of applied medicinal research in health and disease. This dissertation is concerned with the essential role synaptic vesicles play in neurotransmission. The studies to be described began at a time when most of the neuroscience world supported the hypothesis that synaptic vesicles are the source of stimulation-released neurotransmitter which produces the subsequent transynaptic signal transduction. The storage of acetylcholine by vesicles is a critical obligatory step in the normal functioning of cholinergic neurons. Furthermore, potentiation of central cholinergic function, i.e. acetylcholine release, could ameliorate the cognitive impairment associated with the degeneration of cholinergic neurons in diseases such as Alzheimer's (Roberts and Lazareno, 1989). Progress on understanding the biochemistry of acetylcholine storage is being made using the purely cholinergic vesicles which can be isolated in milligram quantities and high purity from the electric organ of the ray Torpedo californica. Vesicles isolated from this organ exhibit noncooperative and homogeneous transport of acetylcholine which is stimulated by adenosine 5{dollar}spprime{dollar}- triphosphate (ATP) and magnesium. A receptor for 1-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) exists on the vesicles with a density of 12 to 16 sites per vesicle. When occupied, the receptor reversibly blocks acetylcholine transport using an apparent noncompetitive mechanism, without any concomitant effect on storage of endogenous acetylcholine. The receptor exhibits enantioselective high affinity for tritium labeled vesamicol, which dissociates in a first order manner. Characterization of vesamicol analogues led to the discovery of many valuable biochemical tools that help prove vesamicol is not acting as an acetylcholine analogue but rather through a complex inhibitory mechanism. Acetylcholine transport activity and vesamicol's affinity and cooperative action display a dependence on the synaptic vesicle concentration, suggesting that a possible dissociable factor is of regulatory importance to the transport system and neurotransmission. The vesamicol receptor purified from Torpedo vesicles shows retention of its characteristic pharmacology, which includes a low affinity acetylcholine antagonism of vesamicol binding. The purified receptor appears to be a proteoglycan of approximately 240,000 M{dollar}sb{lcub}rm r{rcub}{dollar}, whose binding site is conformationally linked to an acetylcholine binding site. The SV1 vesicle-specific antigenic determinant (Kennedy, 1985) copurifies with the vesamicol receptor. Moreover, a monoclonal antibody to SV1 (tor70 or 5G1; Carlson and Kelly, 1983) precipitates a proteoglycan-like vesicle component bearing binding activities for both vesamicol and an acetylcholine analogue. In conclusion, the vesamicol receptor from cholinergic synaptic vesicles is a prominent membrane-spanning proteoglycan. The receptor contains the SV1 epitope and is probably the acetylcholine transporter.