Muscarinic acetylcholine receptors (mAChRs) belong to the superfamily of G-protein-coupled plasma membrane receptors. They regulate a large number of important physiological functions, including the control of neurotransmitter release in the central and peripheral nervous system (Caulfied, 1993; Parnas et al. 2000). Five subtypes of cloned mAChRs are expressed in mammals and are subdivided into two functional groups: the M₁, M₃ and M₅ subtypes which preferentially couple to pertussis toxin (PTX)-insensitive G-proteins of the G_q/11 family, and the M₂ and M₄ subtypes which predominantly couple to PTX-sensitive G-proteins of the G_i/G_o family (Felder, 1995). The subtype of presynaptic mAChRs, as well as the mechanism underlying the regulation of acetylcholine (ACh) release at the skeletal neuromuscular junction (NMJ) are still largely unknown.

In the present study we used subtype-specific antibodies and Western blotting of lysates obtained both from innervated and non-innervated regions of the mouse diaphragm to directly demonstrate that mammalian neuromuscular junctions express mAChR subtypes M₁ to M₄, and that localisation of all subtypes is highly restricted to the innervated part of the muscle. To elucidate the roles of the mAChR subtypes in regulating ACh release evoked by the presynaptic action potential, we evaluated the quantal content of endplate potentials by the method of failures on isolated phrenic-hemidiaphragm preparations removed from adult normal and collagen Q-deficient mice killed by dislocation of the cervical vertebrae.

Muscarine decreased evoked ACh release by 50 ± 12 % (mean ± S.D., n = 9; P < 0.05, Student’s paired t test) in normal NMJs, but it increased ACh release (40 ± 10% n = 4; P < 0.03) both in collagen Q-deficient NMJs completely lacking acetylcholinesterase (AChE) (Feng et al. 1999), and in normal junctions (17 ± 6% n = 9; P < 0.05) when AChE was inhibited by the specific inhibitor fasciculin-2. The muscarine-elicited depression of ACh release in normal NMJs was completely abolished by pretreatment with PTX or methoctramine, a selective M₂ antagonist, but was not affected by M₄-selective muscarinic toxin MT-3. These results indicate the involvement of the M₂ mAChR in decreasing ACh release. The muscarine-induced increase of ACh release in AChE-deficient NMJs was not affected by PTX, but was completely blocked by a specific M₁ antagonist MT-7. Our results show that the M₁ and M₂ mAChRs have opposite presynaptic functions in modulating ACh release, and that regulation of transmitter release by the two receptor subtypes depends on the functional state of AChE at the NMJ.This work was funded in part by AFM. J. Minic was supported by a fellowship from AFM.

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