Acetylcholine synthesis and quantal release reconstituted by transfection of mediatophore and choline acetyltranferase cDNAs

Neuroblastoma N18TG-2 cells cannot synthesize or release acetylcholine (ACh ), and do not express proteins involved in transmitter storage and vesicle fusion. We restored some of these functions by transfecting N18TG-2 cells w ith cDNAs of either rat choline acetyltransferase (ChAT), or Torpedo mediat ophore 16-kDa subunit, or both. Cells transfected only with ChAT synthesize d but did not release ACh. Cells transfected only with mediatophore express ed Ca2+-dependent ACh release provided they were previously filled with the transmitter. Cell lines produced after cotransfection of ChAT and mediatop hore cDNAs released the ACh that was endogenously synthesized. Synaptic-lik e vesicles were found neither in native N18TG-2 cells nor in ChAT-mediatoph ore cotransfected clones, where all the ACh content was apparently cytosoli c. Furthermore, restoration of release did not result from enhanced ACh acc umulation in intracellular organelles consecutive to enhanced acidification by V-ATPase, as Torpedo 16 kDa transfection did not increase, but decrease d the V-ATPase-driven proton transport. Using ACh-sensitive Xenopus myocyte s for real-time recording of evoked release, we found that cotransfected ce lls released ACh in a quantal manner. We compared the quanta produced by Ch AT-mediatophore cotransfected clones to those produced by clones transfecte d with mediatophore alone (artificially filled with ACh). The time characte ristics and quantal size of currents generated in the myocyte were the same in both conditions. However, cotransfected cells released a larger proport ion of their initial ACh store. Hence, expression of mediatophore at the pl asma membrane seems to be necessary for quantal ACh release; the process wo rks more efficiently when ChAT is operating as well, suggesting a functiona l coupling between ACh synthesis and release.