Thiochrome Enhances Acetylcholine Affinity at Muscarinic M4 Receptors: Receptor Subtype Selectivity via Cooperativity Rather than Affinity

Thiochrome (2,7-dimethyl-5 H -thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of l -[ 3 H] N -methyl scopolamine ([ 3 H]NMS) to the five human muscarinic receptor subtypes (M 1 –M 5 ) at concentrations up to 0.3 mM. In contrast, it inhibits [ 3 H]NMS dissociation from M 1 to M 4 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 [ 3 H]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 [ 3 H]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 [ 3 H]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-[ 35 S]thiotriphosphate) ([ 35 S]GTPγS) 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 [ 3 H]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.