Calcium-dependent acetylcholine release from Xenopus oocytes: simultaneous ionic currents and acetylcholine release recordings.

Abstract

The fusion of synaptic vesicles with presynaptic membranes is controlled by a complex network of protein-protein and protein-lipid interactions. SNAP-25, syntaxin and synaptobrevin (SNARE complex) are thought to participate in the formation of the core of the membrane fusion machine but the molecular basis of SNARE interactions is not completely understood. Thus, it would be interesting to design experiments to test those relationships in a new model. Xenopus laevis oocytes are valuable tools for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Here we show that SNARE proteins are present in native Xenopus oocytes and that those oocytes injected with acetylcholine and presynaptic plasma membranes extracted from the electric organ of Torpedo marmorata assume some of the functions of a cholinergic nerve terminal. Neurotransmitter release and macroscopic currents were recorded and analysed simultaneously in a single oocyte electrically depolarized: acetylcholine release was detected using a chemiluminiscent method and calcium entry was measured by exploiting the endogenous Ca2+-activated chloride current of the oocyte with a two-electrode voltage-clamp system. Neurotransmitter release was calcium- and voltage-dependent and partially reduced in the presence of several calcium channel blockers. Clostridial neurotoxins, both holotoxin and injected light-chain forms, also inhibited acetylcholine release. We also studied the role of the SNARE complex in synaptic transmission and membrane currents by using monoclonal antibodies against SNAP-25, syntaxin or VAMP/synaptobrevin. The use of antibodies against VAMP/synaptobrevin, SNAP-25 and syntaxin inhibited acetylcholine release, as did clostridial toxins. However, macroscopic currents were only modified either by syntaxin antibody or by Botulinium-C1 neurotoxin. This model constitutes a new approach for understanding the vesicle exocytosis processes.