Use of knockout mice reveals involvement of M₂-muscarinic receptors in control of the kinetics of acetylcholine release

We have previously suggested that presynaptic M₂-muscarinic receptors (M₂R) 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 M₂R (M₂-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 Ca²⁺ 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 M₂-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 [Ca²⁺]_o or [Mg²⁺]_o in M₂-KO mice but not in WT mice. Measurements of Ca²⁺ currents revealed that the shorter rise time of the postsynaptic current seen in high [Mg²⁺]_o in M₂-KO mice was not produced by a shorter wave of the presynaptic Ca²⁺ current. These results support our earlier findings and provide direct evidence for the major role that presynaptic M₂-muscarinic receptors play in the control of the time course of evoked acetylcholine release under physiological conditions.