Quantal acetylcholine release induced by mediatophore transfection.

Abstract

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...

Quantal transmitter release by glioma cells: quantification of intramembrane particle changes

“…This is much less than the 6000^10 000 molecules estimated for the full quantum in adult neuromuscular or nerve^electroplaque junctions (Ku¥er and Yoshikami, 1975;Girod et al, 1993), but compares well with the values reported in embryonic endplates (Evers et al, 1989), sympathetic ganglia (Bennett, 1995), or in the population of small miniature currents which is believed to be the substructure of the classical quantum (Kriebel and Gross, 1974;Girod et al, 1993). A slightly larger value (200 pA) was obtained using neuroblastoma N18-TG-2 cells after transfection with the mediatophore, a proteolipid present in the presynaptic membrane cholinergic nerve terminals (Falk-Vairant et al, 1996a).…”

“…Moreover, IMPs were observed during ACh release in the membrane of proteoliposomes containing mediatophore as the unique protein (Brochier et al, 1992). Also, transfection of neuroblastoma N18-TG-2 cells with mediatophore cDNA induced both a Ca 2þ -dependent and quantal ACh release (Falk-Vairant et al, 1996a;, and a transient increase of medium and large IMPs , as summarised in Table 1. The 15-kDa proteolipid forming mediatophore is implicated in several cellular processes. It is a component of the membrane sector of V-ATPase (Nelson and Harvey, 1999); it was found in invertebrate gap junctions (Finbow and Pitts, 1998) and was recently identi¢ed in the yeast as the Ca 2þ -sensitive proteolipid involved in membrane fusion (Peters et al, 2001).…”

The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles inParamecium

“…Apart from its enzymatic action as a proton pump, the V 0 -subcomplex of the V-ATPase was found to be localized in the plasma membrane and to be involved in the release of neurotransmitter in mammalian cells (Falk-Vairant et al, 1996;Morel, 2003). A role of the V 0 -sector was also shown in homotypic membrane fusion of vacuoles in yeast (Peters et al, 2001).…”

“…Most of the V 1 -domains dissociate from their V 0 counterparts at vacuoles in yeast when glucose is depleted in the medium (Kane and Smardon, 2003). Whether other physiological conditions exist that lead to a dissociation of the enzyme is unclear.Apart from its enzymatic action as a proton pump, the V 0 -subcomplex of the V-ATPase was found to be localized in the plasma membrane and to be involved in the release of neurotransmitter in mammalian cells (Falk-Vairant et al, 1996;Morel, 2003). A role of the V 0 -sector was also shown in homotypic membrane fusion of vacuoles in yeast (Peters et al, 2001).…”

The vacuolar H+-ATPase: a universal proton pump of eukaryotes

“…Indeed, reconstituted subunit c in liposomes is able to form an oligomeric complex that is permeable to acetylcholine and glutamate [116,137]. The question of mediatophore and neurotransmitter release has proved as controversial as ductin and gap junctions, although a recent study has shown that quantal release of acetylcholine can be achieved when subunit c is present in the cell membrane [138]. It has been suggested that subunit c could be part of the postulated ' fusion pore ' complex, which has the potential to provide an efficient pathway for neurotransmitter release from synaptic vesicles [123,136].…”