Functional Activity of the M₂ and M₄ Receptor Subtypes in the Spinal Cord Studied with Muscarinic Acetylcholine Receptor Knockout Mice

Abstract

Stimulation of spinal muscarinic acetylcholine receptors (mAChRs) produces potent analgesia. Both M₂ and M₄ mAChRs are coupled to similar G proteins (G_i/o family) and play a critical role in the analgesic action of mAChR agonists. To determine the relative contribution of M₂ and M₄ subtypes to activation of G_i/o proteins in the spinal cord, we examined the receptor-mediated guanosine 5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in M₂ and M₄ subtype knockout (KO) mice. Basal [³⁵S]GTPγS binding in the spinal cord was similar in the wild-type controls, M₂ and M₄ single-KO, and M₂/M₄ double-KO mice. The spinal [³⁵S]GTPγS binding stimulated by either muscarine or oxotremorine-M was not significantly different among three groups of wild-type mouse strains. In M₂ single-KO and M₂/M₄ double-KO mice, the agonist-stimulated [³⁵S]GTPγS binding was completely abolished in the spinal cord. Furthermore, the agonist-stimulated [³⁵S]GTPγS binding in the spinal cord of M₄ single-KO mice was significantly reduced (∼15%), compared with that in wild-type controls. On the other hand, the spinal [³⁵S]GTPγS binding stimulated by a μ-opioid agonist was not significantly different between wild-type and M₂ and M₄ KO mice. This study provides complementary new evidence that M₂ is the most predominant mAChR subtype coupled to the G_i/o proteins in the spinal cord. Furthermore, these data suggest that a small but functionally significant population of M₄ receptors exists in the mouse spinal cord. The functional activity of these M₄ receptors seems to require the presence of M₂ receptors.