Solubilization and Partial Purification of a Presynaptic Membrane Protein Ensuring Calcium-Dependent Acetylcholine Release from Proteoliposomes

In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.