Acetylcholine release and the cholinergic genomic locus.

Choline acetyltransferase and vesicular acetylcholine-transporter genes are adjacent and coregulated. They define a cholinergic locus that can be turned on under the control of several factors, including the neurotrophins and the cytokines. Hirschprung's disease, or congenital megacolon, is characterized by agenesis of intramural cholinergic ganglia in the colorectal region. It results from mutations of the RET (GDNF-activated) and the endothelin-receptor genes, causing a disregulation in the cholinergic locus. Using cultured cells, it was shown that the cholinergic locus and the proteins involved in acetylcholine (ACh) release can be expressed separately ACh release could be demonstrated by means of biochemical and electrophysiological assays even in noncholinergic cells following preloading with the transmitter. Some noncholinergic or even nonneuronal cell types were found to be capable of releasing ACh quanta. In contrast, other cells were incompetent for ACh release. Among them, neuroblastoma N18TG-2 cells were rendered release-competent by transfection with the mediatophore gene. Mediatophore is an ACh-translocating protein that has been purified from plasma membranes of Torpedo nerve terminal; it confers a specificity for ACh to the release process. The mediatophores are activated by Ca2+; but with a slower time course, they can be desensitized by Ca2+. A strictly regulated calcium microdomain controls the synchronized release of ACh quanta at the active zone. In addition to ACh and ATP, synaptic vesicles have an ATP-dependent Ca2+ uptake system; they transiently accumulate Ca2+ after a brief period of stimulation. Those vesicles that are docked close to Ca2+ channels are therefore in the best position to control the profile and dynamics of the Ca2+ microdomains. Thus, vesicles and their whole set of associated proteins (SNAREs and others) are essential for the regulation of the release mechanism in which the mediatophore seems to play a key role.