Abstract

Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of l-[³H]N-methyl scopolamine ([³H]NMS) to the five human muscarinic receptor subtypes (M₁–M₅) at concentrations up to 0.3 mM. In contrast, it inhibits [³H]NMS dissociation from M₁ to M₄ receptors at submillimolar concentrations and from M₅ receptors at 1 mM. These results suggest that thiochrome binds allosterically to muscarinic receptors and has approximately neutral cooperativity with [³H]NMS at M₁ to M₄ and possibly M₅ receptors. Thiochrome increases the affinity of acetylcholine (ACh) 3- to 5-fold for inhibiting [³H]NMS binding to M₄ receptors but has no effect on ACh affinity at M₁ to M₃ or M₅ receptors. Thiochrome (0.1 mM) also increases the direct binding of [³H]ACh to M₄ receptors but decreases it slightly at M₂ receptors. In agreement with the binding data, thiochrome does not affect the potency of ACh for stimulating the binding of guanosine 5′-O-(3-[³⁵S]thiotriphosphate) ([³⁵S]GTPγS) to membranes containing M₁ to M₃ receptors, but it increases ACh potency 3.5-fold at M₄ receptors. It also selectively reduces the release of [³H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M₄ receptors, but not from hippocampal slices, which contain presynaptic M₂ receptors. We conclude that thiochrome is a selective M₄ muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M₁ to M₃ receptors; it therefore demonstrates a powerful new form of selectivity, “absolute subtype selectivity”, which is derived from cooperativity rather than from affinity.