Mediatophore is a protein of "200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.We have previously purified from the nerve terminal membranes of Torpedo electric organ a protein of "200 kDa able to translocate acetylcholine (AcCh) upon the action of calcium (1, 2). The protein, named mediatophore, was localized at the active zones (3) and shown to be a homooligomer of a 16-kDa subunit. The sequence of the subunit was found to be homologous to the proteolipid present in the membrane sector of the V ATPase (4, 5) and also in gap junction proteins (6). An antisense probe to the 16-kDa messenger selectively inhibited expression of the 16-kDa mediatophore subunit and suppressed AcCh release from Xenopus oocytes that were rendered able to synthesize and release AcCh after being injected with electric lobe mRNAs (7,8). These experiments together with pharmacological, morphological, and biochemical observations suggested that mediatophore might indeed be involved in the final step of release-i.e., the translocation of AcCh through the nerve terminal plasma membrane (9, 10). The protein would form a pore, switched on by calcium and capable of releasing AcCh either from the cytoplasm or from vesicular stores (fusion pore). It was therefore crucial to determine whether mediatophore would by itself render cells able to release AcCh and whether such a release would display quantal properties as is the case for physiological transmission of nerve impulses.Experiments along this line were difficult to plan, because the 16-kDa mediatophore subunit is also found in the VATPase, which is involved in storage processes, including the storage of AcCh in vesicles. Indeed, the vesicular AcCh transporter carries the transmitter in exchange to protons accumulated by V-ATPase (11, 12). The problem was simplified when we found that the NG108-15 cells, which result from fusion of N18TG-2 and C6BU-1 cells, had high levels of the 16-kDa protein in their membrane. The 16-kDa protein is also abundant in the membranes of C6BU-1 cells but very low in those of N18TG-2 cells (13). It was also found that NG108-15 and C6BU-1 cells release AcCh after being loaded with transmitter, while N18TG-2 cells were unable to release (13,14). In parallel work (15), the same cell types were transfected with a plasmid encoding choline acetyltransferase and were studied in coculture with myocytes. As synapses were formed, miniature end-plate potentials were recorded from NG108-15...

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