Molecular mechanisms that control initiation and termination of physiological depolarization-evoked transmitter release

Ca2+ is essential for physiological depolarization-evoked synchronous neurotransmitter release. But,whether Ca2+ influxoranother factor controls release initiation is still under debate. The time course of ACh release is controlled by a presynaptic inhibitory G protein-coupled autoreceptor (GPCR), whose agonist-binding affinity is voltage-sensitive. However, the relevance of this property for release control is not known. To resolve this question, we used pertussis toxin (PTX), which uncouples GPCR from its G(i/o) and in turn reduces the affinity of GPCR toward its agonist. We show that IPTX enhances ACh and glutamate release (in mice and crayfish, respectively) and, most importantly, alters the time course of release without affecting Ca2+ currents. These effects are not mediated by G(beta gamma) because its microinjection into the presynaptic terminal did not alter the time course of release. Also, PTX reduces the association of the GPCR with the exocytotic machinery, and this association is restored by the addition of agonist. We offer the following mechanism for control of initiation and termination of physiological depolarization-evoked transmitter release. At rest, release is under tonic block achieved by the transmitter-bound high-affinity presynaptic GPCR interacting with the exocytotic machinery. Upon depolarization, the GPCR uncouples from its G protein and consequently shifts to a low-affinity state toward the transmitter. The transmitter dissociates, the unbound GlPCR detaches from the exocytotic machinery, and the tonic block is alleviated. The free machinery, together with Ca2+ that had already entered, initiates release. Release terminates when the reverse occurs upon repolarization.