We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M 4 muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligandbinding studies revealed that LY2033298 displayed a preference for the active state of the M 4 mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [ 35 S]GTPgS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3b phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M 4 mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [ 3 H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M 4 mAChR knockout mice, validating the M 4 mAChR as a key target of action of this novel allosteric ligand.

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1 The present study examined the eect of a range of doses of chronic nicotine (0.75, 1.5, 3.0 and 30.0 mg kg 71 day 71, s.c., 14 days) upon striatal dopaminergic nerve terminal survival following 6-hydroxydopamine (6-OHDA; 10 mg intrastriatal unilaterally) in rats; and the eects of acute nicotine (1 ) nicotine doses signi®cantly inhibited 6-OHDA-induced degeneration. 4 In wild-type mice, acute nicotine treatment produced signi®cant inhibition of methamphetamineinduced neurodegeneration. In a4 nAChR subunit knockout mice, acute nicotine treatment failed to inhibit methamphetamine-induced neurodegeneration. 5 Nicotine is capable of protecting dopaminergic neurons against Parkinsonian-like neurodegeneration in vivo. In rats, this neuroprotective eect is critically dependent upon nicotine dose and is consistent with the activation of nAChRs, as high, desensitizing doses of nicotine fail to be neuroprotective. Further, neuroprotection is absent in a4 nAChR subunit knockout mice. The current results therefore suggest that activation of a4 subunit containing nAChRs constitutes a major component of the neuroprotective eect of nicotine upon Parkinsonian-like damage in vivo.

The M 4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y 113 C/A 203 G in the M 4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M 4 DREADD with a range of agonists, antagonists, and the recently discovered M 4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3 H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M 4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M 4 DREADD and highlight a role for residues Tyr 113 and Ala 203 in the transmission of cooperativity.The muscarinic acetylcholine receptors (mAChRs) are members of the G protein-coupled receptor (GPCR) superfamily and mediate the majority of the actions of acetylcholine (ACh), in both the central nervous system (CNS) and the periphery (Wess et al., 2003). Within the CNS, the M 1 and M 4 mAChRs show a higher level of expression than the M 2 , M 3 , and M 5 subtypes and have been implicated in the regulation of cognition, sensory processing, motor coordination, and attention (Wess et al., 2007). However, these mAChRs are also characterized by a very high degree of sequence conservation

Accumulation of the amyloid protein (Ab) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism by which Ab exerts its neurotoxic effect is largely unknown. It has been suggested that the peptide can bind to the a7 nicotinic acetylcholine receptor (a7nAChR). In this study, we examined the binding of Ab1-42 to endogenous and recombinantly expressed a7nAChRs. Ab1-42 did neither inhibit the specific binding of a7nAChR ligands to rat brain homogenate or slice preparations, nor did it influence the activity of a7nAChRs expressed in Xenopus oocytes. Similarly, Ab1-42 did not compete for a-bungarotoxin-binding sites on SH-SY5Y cells stably expressing a7nAChRs. The effect of the Ab1-42 on tau phosphorylation was also examined. Although Ab1-42 altered tau phosphorylation in a7nAChR-transfected SH-SY5Y cells, the effect of the peptide was unrelated to a7nAChR expression or activity. Binding studies using surface plasmon resonance indicated that the majority of the Ab bound to membrane lipid, rather than to a protein component. Fluorescence anisotropy experiments indicated that Ab may disrupt membrane lipid structure or fluidity. We conclude that the effects of Ab are unlikely to be mediated by direct binding to the a7nAChR. Instead, we speculate that Ab may exert its effects by altering the packing of lipids within the plasma membrane, which could, in turn, influence the function of a variety of receptors and channels on the cell surface.