Depolarization Initiates Phasic Acetylcholine Release by Relief of a Tonic Block Imposed by Presynaptic M₂ Muscarinic Receptors

Abstract

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...

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

“…M 2 R controls the time course (21) of ACh release without affecting Ca 2ϩ currents (6)(7)(8). To examine whether the voltage-dependent affinity of M 2 R is relevant for release control, we checked for effects of PTX on various aspects of ACh release in the mouse NMJ.…”

“…In ACh release, depolarization plays a dual role: (i) opening Ca 2ϩ channels (fast) and (ii) relief of the ACh-bound M 2 R-imposed tonic block (slightly slower) (3,6). To reveal the latter, the nerve terminal was depolarized to different levels at fixed [Ca 2ϩ ] o , and the relationship between m and the amplitude of the depolarizing pulse (PA) (expressed as the slope of [log m/log PA]) was measured under control conditions and when the M 2 R-imposed tonic block was eliminated by addition of an antagonist or by administration of a strong but very brief depolarizing prepulse (see SI Methods) preceding test pulses of variable amplitudes (6). In both cases, the slope of [log m/log PA] was reduced, suggesting that under physiological conditions depolarization plays the two roles.…”

“…If so, then the slower of the two, relief of the tonic block, governs release initiation. Only when the tonic block does not exist does depolarization fulfill the first role alone, opening Ca 2ϩ channels (6).…”

“…Much of this suggested mechanism was supported experimentally (3)(4)(5)(6)(7)(8) by using mainly the cholinergic neuromuscular junction (NMJ), where the M 2 muscarinic autoreceptor (M 2 R) controls both slow feedback inhibition (9,10) and fast ACh release (6)(7)(8). However, the relevance of this hypothesis for other NTs was not investigated.…”

Voltage regulates adrenergic receptor function

“…Indeed, voltage-dependent binding of neurotransmitters to their receptors, or at least voltage-dependent regulation of their downstream signal, has been described for a few other members of the GPCR family, specifically for M 1 and M 2 muscarinic receptors (4), as well as P 2 Y 1 purinergic receptors (5,6) and metabotropic glutamate receptors (7). Voltage-dependence of GPCRs has been implicated in fine-tuning of neurotransmitter release (8,9), in regulation of cardiac excitability (10), and in potentiation of IP 3 dependent intracellular Ca 2+ signals (6). So far the mechanism underlying this voltage sensitivity of GPCRs is not very well understood; however, the current view is that allosteric regulation of receptor conformations by docking of the G protein seems to be required for voltage sensitivity.…”