Vladimir Dolezal

Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M1 and M2 muscarinic acetylcholine receptors

Xanomeline is a functionally selective M1/M4 muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M1 receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M1 and M2 receptors expressed in Chinese hamster ovary cells and [3H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M2 receptor with similar affinity as at the M1 receptor (KI = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M2 receptors was markedly slower than to M1 receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5′-O-(3-[35S]thio)triphosphate binding at M1 receptors, whereas at M2 receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation for 1 h. Development of xanomeline agonistic effects at the M2 receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M1 than at M2 receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time- and concentration-dependent and vary between the M1 and the M2 receptor.

The transcriptional repressor REST is a critical regulator of the neurosecretory phenotype.

Release of distinct cellular cargoes in response to specific stimuli is a process fundamental to all higher eukaryotes and controlled by the regulated secretory pathway (RSP). However, the mechanism by which genes involved in the RSP are selectively expressed, leading to the establishment and appropriate functioning of regulated secretion remaining largely unknown. Using the rat pheochromocytoma cell line PC12, we provide evidence that, by controlling expression of many genes involved in the RSP, the transcriptional repressor REST can regulate this pathway and hence the neurosecretory phenotype. Introduction of REST transgenes into PC12 cells leads to the repression of many genes, the products of which are involved in regulated secretion. Moreover, chromatin immunoprecipitation assays show that many of the repressed genes recruit the recombinant REST protein to RE1 sites within their promoters and abrogation of REST function leads to reactivation of these transcripts. In addition to the observed transcriptional effects, PC12 cells expressing REST have fewer secretory granules and a reduction in the ability to store and release noradrenaline. Furthermore, an important trigger for synaptic release, influx of calcium through voltage‐operated calcium channels, is compromised. This is the first demonstration of a transcription factor that directly controls expression of many major components of the RSP and provides further insight into the function of REST.

Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors.

Iodinated [125I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ‐specific binding was monitored. Relatively high levels of [125I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [125I]WTX‐binding sites. Specific [125I]WTX binding was partially inhibited by both α‐cobratoxin, a blocker of the α7 and muscle‐type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a Kd of 2.0 ± 0.8 μm and was inhibited by α‐cobratoxin but not by a short‐chain α‐neurotoxin antagonist of the muscle‐type nAChR, suggesting a specific interaction with the α7‐type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 μm did not inhibit binding of [3H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [3H]N‐methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G‐protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G‐proteins with the M1, M4, and M5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs.

A specific multi‐nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro

J. Neurochem. (2012) 120, 631–640.

Multiple promoters drive tissue-specific expression of the human M2 muscarinic acetylcholine receptor gene

Despite the wealth of information on the functional and pharmacological properties of the M2 muscarinic receptor, we know relatively little of structure and regulation of the M2 receptor gene. Here, we describe the organisation of the human M2 gene and its promoters. Four exons are present in the 5′ untranslated region of the human M2 mRNA distributed over 146 kb on chromosome 7 which produce eight different splice variants in the IMR‐32 neuroblastoma cell line. The unexpectedly large size of this gene indicates that transcription initiates much further upstream of the coding region than earlier studies had indicated. We present evidence that there are three distinct human M2 promoters. Analysis of endogenous transcripts revealed that promoter 2 is preferentially used in neuroblastoma cells, whereas promoter 1 in cardiac cells. All promoters are highly conserved across human, mouse, rat and pig. They contain multiple start sites and none possess a TATA‐box. In addition, we describe another M2 promoter that is specific for rat. We show that GATA‐4 transcription factor binds to two sites within the regulatory regions of the M2 gene using reporter gene assays, electromobility shift assays and mutational analysis.

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors.

Background: Cobras “three-finger” nonconventional toxin WTX allosterically modulates muscarinic receptors (mAChRs). Results: Activity of several WTX mutants was analyzed; toxin spatial structure and dynamics were determined; and complexes of toxin with M1 and M3 mAChRs were modeled. Conclusion: Flexible loop II is the major determinant for toxin binding to different mAChRs. Significance: Structural framework for rationalization of target-specific positive/negative allosteric regulation of mAChRs is provided. Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional “three-finger” snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by “three-finger” snake neurotoxins.

Structural Insight into Specificity of Interactions between Non-conventional Three-Finger Toxin WTX and Muscarinic Acetylcholine Receptors

Background: Cobras “three-finger” nonconventional toxin WTX allosterically modulates muscarinic receptors (mAChRs). Results: Activity of several WTX mutants was analyzed; toxin spatial structure and dynamics were determined; and complexes of toxin with M1 and M3 mAChRs were modeled. Conclusion: Flexible loop II is the major determinant for toxin binding to different mAChRs. Significance: Structural framework for rationalization of target-specific positive/negative allosteric regulation of mAChRs is provided. Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional “three-finger” snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by “three-finger” snake neurotoxins.

Lipid-based diets improve muscarinic neurotransmission in the hippocampus of transgenic APPSwe/PS1dE9 mice

Transgenic APPswe/PS1dE9 mice modeling Alzheimers disease demonstrate ongoing accumulation of β-amyloid fragments resulting in formation of amyloid plaques that starts at the age of 4-5 months. Buildup of β-amyloid fragments is accompanied by impairment of muscarinic transmission that becomes detectable at this age, well before the appearance of cognitive deficits that manifest around the age of 12 months. We have recently demonstrated that long-term feeding of trangenic mice with specific isocaloric fish oil-based diets improves specific behavioral parameters. Now we report on the influence of short-term feeding (3 weeks) of three isocaloric diets supplemented with Fortasyn (containing fish oil and ingredients supporting membrane renewal), the plant sterol stigmasterol together with fish oil, and stigmasterol alone on markers of cholinergic neurotransmission in the hippocampus of 5-month-old transgenic mice and their wild-type littermates. Transgenic mice fed normal diet demostrated increase in ChAT activity and attenuation of carbachol-stimulated GTP-γ(35)S binding compared to wild-type mice. None of the tested diets compared to control diet influenced the activities of ChAT, AChE, BuChE, muscarinic receptor density or carbachol-stimulated GTP-γ(35)S binding in wild-type mice. In contrast, all experimental diets increased the potency of carbachol in stimulating GTP-γ(35)S binding in trangenic mice to the level found in wild-type animals. Only the Fortasyn diet increased markers of cholinergic synapses in transgenic mice. Our data demonstrate that even short-term feeding of transgenic mice with chow containing specific lipid-based dietary supplements can influence markers of cholinergic synapses and rectify impaired muscarinic signal transduction that develops in transgenic mice.

Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

Characterization of the Drosophila adenosine receptor: the effect of adenosine analogs on cAMP signaling in Drosophila cells and their utility for in vivo experiments

J. Neurochem. (2012) 121, 383–395.