Outline of therapeutic interventions with muscarinic receptor-mediated transmission.

Muscarinc receptor-mediated signaling takes part in many physiological functions ranging from complex higher nervous activity to vegetative responses. Specificity of action of the natural muscarinic agonist acetylcholine is effected by action on five muscarinic receptor subtypes with particular tissue and cellular localization, and coupling preference with different G-proteins and their signaling pathways. In addition to physiological roles it is also implicated in pathologic events like promotion of carcinoma cells growth, early pathogenesis of neurodegenerative diseases in the central nervous system like Alzheimer's disease and Parkinson's disease, schizophrenia, intoxications resulting in drug addiction, or overactive bladder in the periphery. All of these disturbances demonstrate involvement of specific muscarinic receptor subtypes and point to the importance to develop selective pharmacotherapeutic interventions. Because of the high homology of the orthosteric binding site of muscarinic receptor subtypes there is virtually no subtype selective agonist that binds to this site. Activation of specific receptor subtypes may be achieved by developing allosteric modulators of acetylcholine binding, since ectopic binding domains on the receptor are less conserved compared to the orthosteric site. Potentiation of the effects of acetylcholine by allosteric modulators would be beneficial in cases where acetylcholine release is reduced due to pathological conditions. When presynaptic function is severely compromised, the utilization of ectopic agonists can be a thinkable solution.

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

BACKGROUND AND PURPOSE: Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH: Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³5S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M2 muscarinic acetylcholine receptor. KEY RESULTS: Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. CONCLUSIONS AND IMPLICATIONS: These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding.