Neuroactive steroids, WIN-compounds and cholesterol share a common binding site on muscarinic acetylcholine receptors.

Endogenous neurosteroids and their synthetic analogues-neuroactive steroids-have been found to bind to muscarinic acetylcholine receptors and allosterically modulate acetylcholine binding and function. Using radioligand binding experiments we investigated their binding mode. We show that neuroactive steroids bind to two binding sites on muscarinic receptors.

Allosteric Modulation of GPCRs of Class A by Cholesterol.

G-protein coupled receptors (GPCRs) are membrane proteins that convey extracellular signals to the cellular milieu. They represent a target for more than 30% of currently marketed drugs. Here we review the effects of membrane cholesterol on the function of GPCRs of Class A.

The operational model of allosteric modulation of pharmacological agonism.

Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivity and bias to activation of specific signalling pathways. The operational model (OM) of pharmacological agonism is a useful means for achieving this goal. Allosteric ligands bind to receptors at sites that are distinct from those of endogenous agonists that interact with the orthosteric domain on the receptor.

Current Advances in Allosteric Modulation of Muscarinic Receptors.

Allosteric modulators are ligands that bind to a site on the receptor that is spatially separated from the orthosteric binding site for the endogenous neurotransmitter. Allosteric modulators modulate the binding affinity, potency, and efficacy of orthosteric ligands. Muscarinic acetylcholine receptors are prototypical allosterically-modulated G-protein-coupled receptors.

Novel M -selective, G -biased agonists of muscarinic acetylcholine receptors.

Background And Purpose: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects.

A randomized controlled pilot study of the University of Minnesota mentoring excellence training academy: A hybrid learning approach to research mentor training.

Introduction: Research mentor training is a valuable professional development activity. Options for training customization (by delivery mode, dosage, content) are needed to address the many critical attributes of effective mentoring relationships and to support mentors in different institutional settings.

Methods: We conducted a pilot randomized controlled trial to evaluate a hybrid mentor training approach consisting of an innovative, 90-minute, self-paced, online module () followed by workshops based on the curriculum.

Analysis of equilibrium binding of an orthosteric tracer and two allosteric modulators.

Allosteric ligands bind to receptors at sites that are distinct from those endogenous agonists and orthosteric pharmacological agents interact with. Both an allosteric and orthosteric ligand bind simultaneously to the receptor to form a ternary complex, where each ligand influences binding affinity of the other to the receptor, either positively or negatively. Allosteric modulators are an intensively studied group of receptor ligands because of their potentially greater selectivity over orthosteric ligands, with the possibility of fine tuning of the effects of endogenous neurotransmitters and hormones.

Applications and limitations of fitting of the operational model to determine relative efficacies of agonists.

Proper determination of agonist efficacy is essential in the assessment of agonist selectivity and signalling bias. Agonist efficacy is a relative term that is dependent on the system in which it is measured, especially being dependent on receptor expression level. The operational model (OM) of functional receptor agonism is a useful means for the determination of agonist functional efficacy using the maximal response to agonist and ratio of agonist functional potency to its equilibrium dissociation constant (K) at the active state of the receptor.

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors.

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding.

Lipid-Based Diets Improve Muscarinic Neurotransmission in the Hippocampus of Transgenic APPswe/PS1dE9 Mice.

Transgenic APPswe/PS1dE9 mice modeling Alzheimer's 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.

Towards predictive docking at aminergic G-protein coupled receptors.

G protein-coupled receptors (GPCRs) are hard to crystallize. However, attempts to predict their structure have boomed as a result of advancements in crystallographic techniques. This trend has allowed computer-aided molecular modeling of GPCRs.

Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors.

Methoctramine (N,N'-bis[6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane] diamine) is an M(2)-selective competitive antagonist of muscarinic acetylcholine receptors and exhibits allosteric properties at high concentrations. To reveal the molecular mechanisms of methoctramine binding and selectivity we took advantage of reciprocal mutations of the M(2) and M(3) receptors in the second and third extracellular loops that are involved in the binding of allosteric ligands. To this end we performed measurements of kinetics of the radiolabeled antagonists N-methylscopolamine (NMS) in the presence of methoctramine and its precursors, fluorescence energy transfer between green fluorescent protein-fused receptors and an Alexa-555-conjugated precursor of methoctramine, and simulation of molecular dynamics of methoctramine association with the receptor.

Changes in Membrane Cholesterol Differentially Influence Preferential and Non-preferential Signaling of the M1 and M3 Muscarinic Acetylcholine Receptors.

We have found earlier that changes in membrane cholesterol content have distinct impact on signaling via the M1, M2, or M3 receptors expressed in CHO cells (CHO-M1 through CHO-M3). Now we investigated whether gradual changes in membrane cholesterol exerts differential effects on coupling of the M1 and M3 muscarinic receptors to preferential signaling pathways through Gq/11 and non-preferential Gs G-proteins signaling. Changes in membrane cholesterol resulted in only marginal alterations of antagonist and agonist affinity of the M1 and M3 receptors, and did not influence precoupling of either subtype.

Uncoupling of M1 muscarinic receptor/G-protein interaction by amyloid β(1-42).

The overproduction of β-amyloid (Aβ) fragments in transgenic APPswe/PS1dE9 mice results in formation of amyloid deposits in the cerebral cortex and hippocampus starting around four months of age and leading to cognitive impairment much later. We have previously found an age and transgene-dependent weakening of muscarinic receptor-mediated transmission that was not present in young (6-10-week-old) animals but preceded both amyloid deposits and cognitive deficits. Now we investigated immediate and prolonged in vitro effects of non-aggregated Aβ(1-42) on coupling of individual muscarinic receptor subtypes expressed in CHO (Chinese hamster ovary) cells and their underlying mechanisms.

Subtype differences in pre-coupling of muscarinic acetylcholine receptors.

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex "collides" with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist.

Allosteric Modulation of Muscarinic Acetylcholine Receptors.

An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine).

Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors.

Background: Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity.

M2, M3, and M4 muscarinic receptors are expressed in the guinea pig gallbladder.

Aim: The identity of muscarinic acetylcholine receptors (mAchR) involved in cholinergic-mediated contraction of the guinea pig gallbladder has been a matter of debate. Different groups have suggested the involvement of M(1), M(2), M(3), or M(4) receptor subtypes in the contraction of this tissue. The objective of this study was to identify the mAchR subtypes expressed in the guinea pig gallbladder by RT-PCR.

Membrane cholesterol content influences binding properties of muscarinic M2 receptors and differentially impacts activation of second messenger pathways.

We investigated the influence of membrane cholesterol content on preferential and non-preferential signaling through the M(2) muscarinic acetylcholine receptor expressed in CHO cells. Cholesterol depletion by 39% significantly decreased the affinity of M(2) receptors for [(3)H]-N-methylscopolamine ([(3)H]-NMS) binding and increased B(max) in intact cells and membranes. Membranes displayed two-affinity agonist binding sites for carbachol and cholesterol depletion doubled the fraction of high-affinity binding sites.