Use of Knockout Mice Reveals Involvement of M₂-Muscarinic Receptors in Control of the Kinetics of Acetylcholine Release

Abstract

We have previously suggested that presynaptic M(2)-muscarinic receptors (M(2)R) are involved in the control of the time course of evoked acetylcholine release in the frog neuromuscular junction. The availability of knockout mice lacking functional M(2)R (M(2)-KO) enabled us to address this issue in a more direct way. Using the phrenic diaphragm preparation, we show that in wild-type (WT) mice experimental manipulations known to affect Ca(2+) entry and removal, greatly affected the amount of acetylcholine released (quantal content). However, the time course of release remained unaltered under all these experimental treatments. On the other hand, in the M(2)-KO mice, similar experimental treatments affected both the quantal content and the time course of release. In general, a larger quantal content was accompanied by a longer duration of release. Similarly, the rise time of the postsynaptic current produced by axon stimulation was sensitive to changes in [Ca(2+)](o) or [Mg(2+)](o) in M(2)-KO mice but not in WT mice. Measurements of Ca(2+) currents revealed that the shorter rise time of the postsynaptic current seen in high [Mg(2+)](o) in M(2)-KO mice was not produced by a shorter wave of the presynaptic Ca(2+) current. These results support our earlier findings and provide direct evidence for the major role that presynaptic M(2)-muscarinic receptors play in the control of the time course of evoked acetylcholine release under physiological conditions.

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

“…For electrophysiology, hemidiaphragm neuromuscular preparations were isolated and submerged in the standard bathing solution (15 Ϯ 1°C) as described in ref. 8. Here and in crayfish, small changes in Ca 2ϩ and Mg 2ϩ concentration (1-3 mM) were not compensated for, TTX was added only in focal depolarization, and pH was adjusted to 7.4 with NaOH.…”

“…Under such conditions, we expect that removal of Ca 2ϩ will determine release termination (3). Indeed, although in PTX-untreated mice the addition of the Ca 2ϩ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) did not shorten release kinetics (3,8), in PTX-treated mice it did shorten release (SI Fig. 9).…”

“…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.…”

“…Because GPCRs inhibit Ca 2ϩ channels in a voltage-dependent manner (13,(31)(32)(33)(34), PTX could potentially enhance ER by abolishing this block. We measured presynaptic Ca 2ϩ currents derived from the excitatory nerve terminal current (ENTC) (7,8,35). Because PTX does not enhance release at high depolarizations ( Fig.…”

“…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.…”