The effect of membrane potential on feedback inhibition of acetylcholine (ACh) release was studied using the frog neuromuscular junction. It was found that membrane potential affects the functional affinity (K(i)) of the presynaptic M2 muscarinic receptor. The K(i) for muscarine shifts from approximately 0.23 microm (at resting potential) to approximately 8 microm (at a high depolarization). Measurements of Ca2+ currents in axon terminals showed that the depolarization-mediated shift in K(i) does not stem from depolarization-dependent changes in Ca2+ influx. Pretreatments with pertussis toxin (PTX) abolished the depolarization-dependent shift in K(i); at all depolarizations K(i) was the same and higher (approximately 32 microm) than before PTX treatment. The inhibitory effect of muscarine on ACh release is produced by two independent mechanisms: a slow, PTX-sensitive process, which prevails at low to medium depolarizations and operates already at low muscarine concentrations, and a fast, PTX-insensitive and voltage-independent process, which requires higher muscarine concentrations. Neither of the two processes involves a reduction in Ca2+ influx.

Parnas, H., I. Slutsky, G. Rashkovan, I. Silman, J. Wess, and I. Parnas. Depolarization initiates phasic acetylcholine release by relief of a tonic block imposed by presynaptic M 2 muscarinic receptors. J Neurophysiol 93: 3257-3269, 2005. First published February 9, 2005 doi:10.1152/jn.01131.2004. The role of presynaptic muscarinic autoreceptors in the initiation of phasic acetylcholine (ACh) release at frog and mouse neuromuscular junctions was studied by measuring the dependency of the amount (m) of ACh release on the level of presynaptic depolarization. Addition of methoctramine (a blocker of M 2 muscarinic receptors), or of acetylcholinesterase (AChE), increased release in a voltage-dependent manner; enhancement of release declined as the depolarizing pulse amplitude increased. In frogs and wild-type mice the slope of log m/log pulse amplitude (PA) was reduced from about 7 in the control to about 4 in the presence of methoctramine or AChE. In M 2 muscarinic receptor knockout mice, the slope of log m/log PA was much smaller (about 4) and was not further reduced by addition of either methoctramine or AChE. The effect of a brief (0.1 ms), but strong (Ϫ1.2 A) depolarizing prepulse on the dependency of m on PA was also studied. The depolarizing prepulse had effects similar to those of methoctramine and AChE. In particular, it enhanced release of test pulses in a voltage-dependent manner and reduced the slope of log m/log PA from about 7 to about 4. Methoctramine ϩ AChE occluded the prepulse effects. In knockout mice, the depolarizing prepulse had no effects. The cumulative results suggest that initiation of phasic ACh release is achieved by depolarization-mediated relief of a tonic block imposed by presynaptic M 2 muscarinic receptors. I N T R O D U C T I O NPresynaptic G-protein-coupled receptors (GPCRs) are known to modulate neurotransmitter release by various mechanisms (MacDermott et al. 1999), one of which involves direct modulation of proteins of the release machinery (for example, Blackmer et al. 2001;Capogna et al. 1996;Scholz and Miller 1992;Silinsky 1984;Trudeau et al. 1996; for review see Miller 1998).We recently proposed that control of the time course of neurotransmitter release, both of initiation and termination, is achieved by a direct effect of presynaptic inhibitory autoreceptors on key proteins of the release machinery . In particular, we proposed that at rest (i.e., at resting potential and resting low level of transmitter in the cleft) the release machinery is under tonic block. The block is achieved as a result of an interaction of the transmitter-occupied inhibitory autoreceptor with the core proteins of the release machinery. At resting potential the receptor is in a high-affinity state and thus can be occupied even at the low tonic concentration of transmitter. Initiation of release is achieved by depolarization shifting the autoreceptor to a low-affinity state, resulting in fast dissociation of the transmitter from the receptor. The free autoreceptor rapidly detaches from the proteins o...

Ca 2؉ is essential for physiological depolarization-evoked synchronous neurotransmitter release. But, whether Ca 2؉ influx or another 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 Gi/o and in turn reduces the affinity of GPCR toward its agonist. We show that PTX enhances ACh and glutamate release (in mice and crayfish, respectively) and, most importantly, alters the time course of release without affecting Ca 2؉ currents. These effects are not mediated by G␤␥ 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 GPCR detaches from the exocytotic machinery, and the tonic block is alleviated. The free machinery, together with Ca 2؉ that had already entered, initiates release. Release terminates when the reverse occurs upon repolarization.G protein-coupled receptor ͉ neurotransmitter release ͉ pertussis toxin ͉ presynaptic receptors C a 2ϩ influx is essential for physiological depolarizationinduced neurotransmitter (NT) release (1, 2). A broader, Ca 2ϩ voltage, hypothesis suggests that two factors control release: Ca 2ϩ and G protein-coupled receptors (GPCRs), whose agonist-binding affinity is voltage-dependent (3). The mechanism suggested for this control is as follows. (i) At resting potential and rest concentration (nMs) of transmitter, the release machinery (SNARE proteins and synaptotagmin) is under tonic block imposed by the transmitter-bound high-affinity (nMs) GPCR. (ii) Depolarization shifts the GPCR to a lowaffinity state ( Ms), resulting in rapid transmitter dissociation (it should be emphasized that at this stage release of NT did not occur yet, and the concentration of NT in the synapse is still in the nM range). (iii) The unbound GPCR detaches from the release machinery to relieve the tonic block. The free-release machinery together with Ca 2ϩ , which had already entered, initiates release. (iv) Upon repolarization, release terminates because the receptor returns to its high-affinity state and the tonic block is reinstated.Much of this suggested mechanism was supported experimentally (3-8) by using mainly the cholinergic neuromuscular junction (N...

Release of excitatory transmitter from boutons on crayfish nerve terminals was inhibited by (R,S)-baclofen, an agonist at GABAB receptors. Baclofen had no postsynaptic actions as it reduced quantal content without affecting quantal amplitude. The effect of baclofen increased with concentration producing 18% inhibition at 10 microM; EC50, 50% inhibition at 30 microM; maximal inhibition, 85% at 100 microM and higher. There was no desensitization, even with 200 or 320 microM baclofen. Phaclofen, an antagonist at GABAB receptors, competitively antagonized the inhibitory action of baclofen (KD = 50 microM, equivalent to a pA2 = 4.3 +/- 0.1). Phaclofen on its own at concentrations below 200 microM had no effect on release, whereas at 200 microM phaclofen itself increased the control level of release by 60%, as did 2-hydroxy-saclofen (200 microM), another antagonist at GABAB receptors. This increase was evidently due to antagonism of a persistent level of GABA in the synaptic cleft, since the effect was abolished by destruction of the presynaptic inhibitory fiber, using intra-axonal pronase. We conclude that presynaptic GABAB receptors, with a pharmacological profile similar to that of mammalian GABAB receptors, are involved in the control of transmitter release at the crayfish neuromuscular junction.