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.

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 μM, ws-LYNX1 competed with (125)I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μM ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μM caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.

Experiments were performed to investigate the effects on the spontaneous, nonquantal release of acetylcholine (AcCho) from motor nerve terminals of substances known to inhibit the AcCho transport system present in cholinergic synaptic vesicles. In mouse diaphragms, the hyperpolarization normally produced by d-tubocurarine in the endplate area of muscle fibers that had been treated by an anticholinesterase was partly or completely blocked by 2-(4-phenylpiperidino)cyclohexanol (AH5183, 0.1-1 microM), quinacrine (0.1 microM), and tetraphenylborate (1 microM). Since the sensitivity of the subsynaptic area to AcCho was not changed, the block of the hyperpolarizing action of d-tubocurarine indicated in inhibition of the spontaneous, nonquantal release of AcCho. This was confirmed in experiments on rat diaphragm using direct radioenzymatic measurement of the AcCho released into the incubation medium. The release of AcCho from the innervated diaphragm was decreased by about 50% in the presence of AH5183 (0.01-1 microM) and by 42% in the presence of quinacrine (0.1-1 microM). The AcCho released was presumably neural, since the release of AcCho from 4-day denervated diaphragms was not diminished by either AH5183 or quinacrine. The results indicate that the spontaneous release of AcCho from the motor nerve terminals is highly sensitive to low concentrations of specific inhibitors and is probably mediated by a carrier. It is proposed that spontaneous release is due to the incorporation into the membrane of the nerve terminal during exocytosis of the vesicular transport system responsible for moving AcCho into the vesicle.

Muscarinic M₂ receptors preferentially couple with the G_i/o class of G-proteins to inhibit cAMP synthesis. However, they can also stimulate net synthesis of cAMP and inositol phosphate (IP) accumulation. We investigated in intact Chinese hamster ovary (CHO) cells expressing human M₂ receptors (CHO-M₂ cells) whether direct interaction of M₂ receptors with G_s and G_q/11 G-proteins is responsible for the latter effects. Suppression of the G_sα subunit using RNA interference abolished stimulation of cAMP synthesis induced by 1 mM carbachol in both control and pertussis toxin-treated CHO-M₂ cells but had no effect on the inhibition of forskolin-stimulated cAMP synthesis. Carbachol stimulated accumulation of IP with an EC₅₀ of 79 μM. Removal of the G_q,G₁₁, or both α subunits reduced this response by 78, 54, and 92%, respectively, whereas suppression of the G_sα subunit had no effect. Similar results obtained in CHO cells expressing M₁ receptors that preferentially couple with G_s and G_q/11 G-proteins confirmed the efficiency of siRNA treatments. Stimulation of M₂ receptors in control and pertussis toxin-treated cells by a series of full agonists with respect to inhibition of adenylyl cyclase displayed different efficacies in stimulating IP accumulation. Carbachol, acetylcholine, and oxotremorine-M [N,N,N-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium] behaved as full agonists, furmethide (N,N,N-trimethyl-2-furanmethammonium) and methylfurmethide [(5-methyl-2-furyl)methyltrimethylammonium] were partial agonists, and oxotremorine (1-[4-(1-pyrrolidinyl)-2-butynyl]-2-pyrrolidinone) had no effect. Our results provide direct evidence of M₂ receptor coupling with the α subunits of G_s and G_q/11 G-proteins and demonstrate induction of multiple receptor conformational states dependent on both the concentration and the nature of the agonist used.