N-Ethylmaleimide differentiates between the M2- and M4-autoreceptor-mediated inhibition of acetylcholine release in the mouse brain.

Muscarinic M2 and M4 receptors resemble each other in brain distribution, function, and Gi/o protein signaling. However, there is evidence from human recombinant receptors that the M4 receptor also couples to Gs protein whereas such an alternative signaling is of minor importance for its M2 counterpart. The question arises whether this property is shared by native receptors, e.g., the murine hippocampal M2- and the striatal M4-autoreceptor. To this end, the electrically evoked tritium overflow was studied in mouse hippocampal and striatal slices pre-incubated with 3H-choline. 3H-Acetylcholine release in either region was inhibited by the potent muscarinic receptor agonist iperoxo (pIC50 8.6-8.8) in an atropine-sensitive manner (apparent pA2 8.6-8.8); iperoxo was much more potent than oxotremorine (pIC50 6.5-6.6). In hippocampal slices, N-ethylmaleimide (NEM) 32 µM, which inactivates Gi/o proteins, tended to shift the concentration-response curve of iperoxo (pIC50 8.8) to the right (pIC50 8.5) and depressed its maximum from 85 to 69%. In striatal slices, the inhibitory effect of iperoxo declined at concentrations higher than 0.1 µM, yielding a biphasic curve with a pIC50 of 8.6 for the falling part and a pEC50 of 6.4 for the rising part of the curve. The inhibitory effect of iperoxo 10 µM (47%) after NEM pre-treatment was lower by about 35% compared to the maximum (74%) obtained without NEM. In conclusion, our data, which need to be confirmed by pertussis toxin, might suggest that in the striatum, unlike the hippocampus, stimulatory Gs protein comes into play at high concentrations of a muscarinic receptor agonist.

Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor.

Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M2-receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor.