Thiochrome enhances acetylcholine affinity at muscarinic M4 receptors:: Receptor subtype selectivity via cooperativity rather than affinity

Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of L-[H-3]N-methyl scopolamine ([H-3]NMS) to the five human muscarinic receptor subtypes (M-1 M-5) at concentrations up to 0.3 mM. In contrast, it inhibits [H-3]NMS dissociation from M-1 to M4 receptors at submillimolar concentrations and from M-5 receptors at 1 mM. These results suggest that thiochrome binds allosterically to muscarinic receptors and has approximately neutral cooperativity with [H-3]NMS at M-1 to M-4 and possibly M-5 receptors. Thiochrome increases the affinity of acetylcholine (ACh) 3- to 5-fold for inhibiting [H-3]NMS binding to M-4 receptors but has no effect on ACh affinity at M-1 to M-3 or M-5 receptors. Thiochrome (0.1 mM) also increases the direct binding of [H-3]ACh to M-4 receptors but decreases it slightly at M-2 receptors. In agreement with the binding data, thiochrome does not affect the potency of ACh for stimulating the binding of guanosine 5'-O-(3-[S-35]thiotriphosphate) ([S-35]GTPgammaS) to membranes containing M-1 to M-3 receptors, but it increases ACh potency 3.5-fold at M-4 receptors. It also selectively reduces the release of [H-3]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M-4 receptors, but not from hippocampal slices, which contain presynaptic M-2 receptors. We conclude that thiochrome is a selective M-4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M-1 to M-3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity.