Vladimír Doležal - Academia.edu (original) (raw)

Papers by Vladimír Doležal

Research paper thumbnail of Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors

Molecular pharmacology, 2014

Methoctramine (N,N'-bis[6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane] diamine) is an M(2)-s... more 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. We confirm the hypothesis that methoctramine high-affinity binding to the M(2) receptors involves simultaneous interaction with both the orth...

Research paper thumbnail of The operational model of allosteric modulation of pharmacological agonism

Scientific Reports

Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivit... more 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. An allosteric modulator and an orthosteric agonist bind simultaneously to the receptor to form a ternary complex, where the allosteric modulator affects the binding affinity and operational efficacy of the agonist. Allosteric modulators are an intensively studied group of receptor ligands because of their selectivity and preservation of physiological space-time pattern of the signals they modulate. We analysed the operational model of allosterically-modulated agonism (OMAM) including modulation by allosteric agonists. Similar to OM, several parameters of OMAM are inter-dependent. We derived equations describing mutual relationships among parameters of the functional response and OMAM. We present a workflow for the robust fitting of OMAM to experimental data using derived equations.

Research paper thumbnail of Agonist-Specific Conformations of the M2 Muscarinic Acetylcholine Receptor Assessed by Molecular Dynamics

Journal of Chemical Information and Modeling

Research paper thumbnail of Novel M 2 ‐selective, G i ‐biased agonists of muscarinic acetylcholine receptors

British Journal of Pharmacology

Research paper thumbnail of Role of membrane cholesterol in differential sensitivity of muscarinic receptor subtypes to persistently bound xanomeline

Research paper thumbnail of Novel long-acting antagonists of muscarinic ACh receptors

British Journal of Pharmacology

The aim of this study was to develop potent and long-acting antagonists of muscarinic ACh recepto... more The aim of this study was to develop potent and long-acting antagonists of muscarinic ACh receptors. The 4-hexyloxy and 4butyloxy derivatives of 1-[2-(4-oxidobenzoyloxy)ethyl]-1,2,3,6-tetrahydropyridin-1-ium were synthesized and tested for biological activity. Antagonists with long-residence time at receptors are therapeutic targets for the treatment of several neurological and psychiatric human diseases. Their long-acting effects allow for reduced daily doses and adverse effects.

Research paper thumbnail of Analysis of equilibrium binding of an orthosteric tracer and two allosteric modulators

PLOS ONE

The derivation of the equations for tracer binding expressed as a ratio of the tracer binding in ... more The derivation of the equations for tracer binding expressed as a ratio of the tracer binding in the presence of allosteric modulator (Y') to the tracer binding in the absence of allosteric modulator (Y) based on equilibrium dissociation constants (K) and factors of cooperativity (Greek letters) shown in main manuscript is described below. These equations are based on the the main manuscript describing interaction of tracer X and two allosteric modulators A and B. The relationship between the concentration of the ligands and their complexes is derived assuming that the reactions take place under pseudo-first order conditions when the ligands are present in sufficient excess over the receptor so that the formation of the complexes leaves the free concentration of ligands virtually unchanged.

Research paper thumbnail of Applications and limitations of fitting of the operational model to determine relative efficacies of agonists

Scientific Reports

Proper determination of agonist efficacy is essential in the assessment of agonist selectivity an... more 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 A ) at the active state of the receptor. However, the functional efficacy parameter τ is interdependent on two other parameters of OM; agonist's K A and the highest response that could be evoked in the system by any stimulus (E MAX ). Thus, fitting of OM to functional response data is a tricky process. In this work we analyse pitfalls of fitting OM to experimental data and propose a rigorous fitting procedure where K A and e MAX are derived from half-efficient concentration of agonist and apparent maximal responses obtained from a series of functional response curves. Subsequently, OM with fixed K A and e MAX is fitted to functional response data to obtain τ. The procedure was verified at M 2 and M 4 muscarinic receptors fused with the G 15 G-protein α-subunit. The procedure, however, is applicable to any receptor-effector system.

Research paper thumbnail of Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

Scientific Reports

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G... more 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. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M 2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

Research paper thumbnail of Chapter 25 The non-quantal release of acetylcholine from motor nerve terminals: comment on its likely size

Progress in Brain Research, 1993

Research paper thumbnail of Apolipoprotein E4 reduces evoked hippocampal acetylcholine release in adult mice

Journal of neurochemistry, Jan 3, 2015

Apolipoprotein E4 (apoE4) is the most prevalent genetic risk factor for Alzheimer's disease (... more Apolipoprotein E4 (apoE4) is the most prevalent genetic risk factor for Alzheimer's disease (AD). We utilized apoE4-targeted replacement mice (approved by the Tel Aviv University Animal Care Committee) to investigate whether cholinergic dysfunction, which increases during aging and is a hallmark of AD, is accentuated by apoE4. This revealed that levels of the presynaptic cholinergic marker, vesicular acetylcholine transporter in the hippocampus and the corresponding electrically evoked release of acetylcholine, are similar in 4-month-old apoE4 and apoE3 mice. Both parameters decrease with age. This decrease is, however, significantly more pronounced in the apoE4 mice. The levels of cholinacetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) were similar in the hippocampus of young apoE4 and apoE3 mice and decreased during aging. For ChAT this decrease was similar in the apoE4 and apoE3 mice, whereas it was more pronouced in the apoE4 mice, rega...

Research paper thumbnail of Towards predictive docking at aminergic G-protein coupled receptors

Journal of Molecular Modeling, 2015

G protein-coupled receptors (GPCRs) are hard to crystallize. However, attempts to predict their s... more 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. We analyzed the performance of four molecular modeling programs in pose evaluation of re-docked antagonists / inverse agonists to 11 original crystal structures of aminergic GPCRs using an induced fit-docking procedure. AutoDock and Glide were used for docking. AutoDock binding energy function, GlideXP, Prime MM-GB/SA, and YASARA binding function were used for pose scoring. Root mean square deviation (RMSD) of the best pose ranged from 0.09 to 1.58 Å, and median RMSD of the top 60 poses ranged from 1.47 to 3.83 Å. However, RMSD of the top pose ranged from 0.13 to 7.33 Å and ranking of the best pose ranged from the 1st to 60th out of 60 poses. Moreover, analysis of ligand-receptor interactions of top poses revealed substantial differences from interactions found in crystallographic structures. Bad ranking of top poses and discrepancies between top docked poses and crystal structures render current simple docking methods unsuitable for predictive modeling of receptor-ligand interactions. Prime MM-GB/SA optimized for 3NY9 by multiple linear regression did not work well at 3NY8 and 3NYA, structures of the same receptor with different ligands. However, 9 of 11 trajectories of molecular dynamics simulations by Desmond of top poses converged with trajectories of crystal structures. Key interactions were properly detected for all structures. This procedure also worked well for cross-docking of tested β2-adrenergic antagonists. Thus, this procedure represents a possible way to predict interactions of antagonists with aminergic GPCRs.

Research paper thumbnail of Utilization of Superfused Cerebral Slices in Probing Muscarinic Receptor Autoregulation of Acetylcholine Release

Research paper thumbnail of Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms

Pharmacological research : the official journal of the Italian Pharmacological Society, Jan 13, 2015

We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand ... more We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished functional responses to both classical and atypical agonists. O...

Research paper thumbnail of The synthesis and release of acetylcholine in normal and denervated rat diaphragms during incubation in vitro

The Journal of physiology, 1983

1. Normal and denervated rat diaphragms and neural (central) and aneural (peripheral) parts of no... more 1. Normal and denervated rat diaphragms and neural (central) and aneural (peripheral) parts of normal diaphragms were incubated under several different conditions likely to affect the metabolism of acetylcholine (ACh), with the aim of discovering specific features of the control of neural and aneural ACh in the muscle. The concentrations of ACh in the tissue and the medium were measured at the end of the incubations using a radioenzymatic assay, and the amount of ACh synthesized during the incubations was calculated by subtracting the initial amount of ACh present in the tissue from that found in the tissue plus the medium at the end of the incubations.2. Confirming earlier results obtained with bioassays, it was found that, in a medium with 5 mM-K(+) and 2.5 mM-Ca(2+), denervated diaphragms released ACh into the medium at a rate equal to 47% of that observed in normal diaphragms; the amount of ACh released from aneural parts of normal diaphragms was 51% of that released from their ...

Research paper thumbnail of Tacrine (Tetrahydroaminoacridine) and the Metabolism of Acetylcholine and Choline

Phospholipids and Signal Transmission, 1993

Research paper thumbnail of Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor

PLoS ONE, 2010

Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at t... more Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M 1 and M 4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of xanomeline on the binding characteristics and function of the M 1 muscarinic receptor. Pretreatment of CHO cells that stably express the M 1 receptor for 1 hr with increasing concentrations of xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed xanomelineinduced inhibition of [ 3 H]NMS binding from monophasic to biphasic. The high-affinity xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound xanomeline developed with a time course that parallels the return of receptor activation by prebound xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of xanomeline, but not its persistent binding at an allosteric site. Furthermore, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization.

Research paper thumbnail of Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors

PLoS ONE, 2011

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities ... more 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. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G q/11 , while even-numbered receptors prefer coupling to G i/o . We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist highaffinity binding conformation. Antibodies directed against the C-terminus of the a-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [ 3 H]-Nmethylscopolamine. Antibodies directed against the C-terminus of the a-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors precouple with their preferential classes of G-proteins, but only M 1 and M 3 receptors also pre-couple with non-preferential G i/o G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.

Research paper thumbnail of Uncoupling of M1 muscarinic receptor/G-protein interaction by amyloid β1–42

Neuropharmacology, 2013

The overproduction of b-amyloid (Ab) fragments in transgenic APPswe/PS1dE9 mice results in format... more The overproduction of b-amyloid (Ab) 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 transgenedependent weakening of muscarinic receptor-mediated transmission that was not present in young (6e10-week-old) animals but preceded both amyloid deposits and cognitive deficits. Now we investigated immediate and prolonged in vitro effects of non-aggregated Ab 1e42 on coupling of individual muscarinic receptor subtypes expressed in CHO (Chinese hamster ovary) cells and their underlying mechanisms. Immediate application of 1 mM Ab 1e42 had no effect on the binding of the muscarinic antagonist N-methylscopolamine or the agonist carbachol. In contrast, 4-day treatment of CHO cells expressing the M1 muscarinic receptor with 100 nM Ab 1e42 significantly changed the binding characteristics of the muscarinic agonist carbachol and reduced the extent of the M1 receptor-stimulated breakdown of phosphatidylinositol while it did not demonstrate overt toxic effects. The treatment had no influence on the expression of either G-proteins or muscarinic receptors. In concert, we found no change in the gene expression of muscarinic receptor subtypes and gene or protein expression of the G s , G q/11 , and G i/o G-proteins in the cerebral cortex of young adult APPswe/PS1dE9 mice that demonstrate high concentrations of soluble Ab 1e42 and impaired muscarinic receptor-mediated G-protein activation. Our results provide strong evidence that the initial injurious effects of Ab 1e42 on M1 muscarinic receptor-mediated transmissionis is due to compromised coupling of the receptor with G q/11 G-protein. Abbreviations: AD, Alzheimer's disease; CHO cells, Chinese hamster ovary cells; CHO-M1 through CHO-M5, Chinese hamster ovary cells expressing individual subtypes (M1 through M5) of human muscarinic receptors. (H. Janí cková), rudajev@biomed.cas.cz (V. Rudajev), zimcik@biomed.cas.cz (P. Zim cík), jakubik@biomed.cas.cz (J. Jakubík), heikki.tanila@uef.fi (H. Tanila), elfak001@umn.edu (E.E. El-Fakahany), dolezal@ biomed.cas.cz (V. Dole zal).

Research paper thumbnail of The Increase of Choline Acetyltransferase Activity by Docosahexaenoic Acid in NG108-15 Cells Grown in Serum-free Medium is Independent of its Effect on Cell Growth

Neurochemical Research, 2006

We investigated the influence of the polyunsaturated docosahexaenoic acid (22:6n-3; DHA) on the c... more We investigated the influence of the polyunsaturated docosahexaenoic acid (22:6n-3; DHA) on the constitutive expression of choline acetyltransferase (ChAT) in native and induced expression in differentiated cholinergic cells NG108-15 grown in serum-free medium. Elimination of serum-derived trophic support resulted in growth arrest and a strong decrease of ChAT activity. In either conditions, DHA largely rescued general indicators of cell growth and function, and partially prevented the decrease of ChAT activity. However, the maximal effect on general cell state in native and differentiated cells, and ChAT activity in native cells, was reached at or below 10 lmol/l of DHA. In contrast, maximal induction of ChAT activity in differentiated cells required about six times higher concentrations of DHA. These data thus demonstrate stimulatory effect of DHA on ChAT activity that is independent of its general cell protective properties.

Research paper thumbnail of Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors

Molecular pharmacology, 2014

Methoctramine (N,N'-bis[6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane] diamine) is an M(2)-s... more 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. We confirm the hypothesis that methoctramine high-affinity binding to the M(2) receptors involves simultaneous interaction with both the orth...

Research paper thumbnail of The operational model of allosteric modulation of pharmacological agonism

Scientific Reports

Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivit... more 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. An allosteric modulator and an orthosteric agonist bind simultaneously to the receptor to form a ternary complex, where the allosteric modulator affects the binding affinity and operational efficacy of the agonist. Allosteric modulators are an intensively studied group of receptor ligands because of their selectivity and preservation of physiological space-time pattern of the signals they modulate. We analysed the operational model of allosterically-modulated agonism (OMAM) including modulation by allosteric agonists. Similar to OM, several parameters of OMAM are inter-dependent. We derived equations describing mutual relationships among parameters of the functional response and OMAM. We present a workflow for the robust fitting of OMAM to experimental data using derived equations.

Research paper thumbnail of Agonist-Specific Conformations of the M2 Muscarinic Acetylcholine Receptor Assessed by Molecular Dynamics

Journal of Chemical Information and Modeling

Research paper thumbnail of Novel M 2 ‐selective, G i ‐biased agonists of muscarinic acetylcholine receptors

British Journal of Pharmacology

Research paper thumbnail of Role of membrane cholesterol in differential sensitivity of muscarinic receptor subtypes to persistently bound xanomeline

Research paper thumbnail of Novel long-acting antagonists of muscarinic ACh receptors

British Journal of Pharmacology

The aim of this study was to develop potent and long-acting antagonists of muscarinic ACh recepto... more The aim of this study was to develop potent and long-acting antagonists of muscarinic ACh receptors. The 4-hexyloxy and 4butyloxy derivatives of 1-[2-(4-oxidobenzoyloxy)ethyl]-1,2,3,6-tetrahydropyridin-1-ium were synthesized and tested for biological activity. Antagonists with long-residence time at receptors are therapeutic targets for the treatment of several neurological and psychiatric human diseases. Their long-acting effects allow for reduced daily doses and adverse effects.

Research paper thumbnail of Analysis of equilibrium binding of an orthosteric tracer and two allosteric modulators

PLOS ONE

The derivation of the equations for tracer binding expressed as a ratio of the tracer binding in ... more The derivation of the equations for tracer binding expressed as a ratio of the tracer binding in the presence of allosteric modulator (Y') to the tracer binding in the absence of allosteric modulator (Y) based on equilibrium dissociation constants (K) and factors of cooperativity (Greek letters) shown in main manuscript is described below. These equations are based on the the main manuscript describing interaction of tracer X and two allosteric modulators A and B. The relationship between the concentration of the ligands and their complexes is derived assuming that the reactions take place under pseudo-first order conditions when the ligands are present in sufficient excess over the receptor so that the formation of the complexes leaves the free concentration of ligands virtually unchanged.

Research paper thumbnail of Applications and limitations of fitting of the operational model to determine relative efficacies of agonists

Scientific Reports

Proper determination of agonist efficacy is essential in the assessment of agonist selectivity an... more 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 A ) at the active state of the receptor. However, the functional efficacy parameter τ is interdependent on two other parameters of OM; agonist's K A and the highest response that could be evoked in the system by any stimulus (E MAX ). Thus, fitting of OM to functional response data is a tricky process. In this work we analyse pitfalls of fitting OM to experimental data and propose a rigorous fitting procedure where K A and e MAX are derived from half-efficient concentration of agonist and apparent maximal responses obtained from a series of functional response curves. Subsequently, OM with fixed K A and e MAX is fitted to functional response data to obtain τ. The procedure was verified at M 2 and M 4 muscarinic receptors fused with the G 15 G-protein α-subunit. The procedure, however, is applicable to any receptor-effector system.

Research paper thumbnail of Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

Scientific Reports

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G... more 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. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M 2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

Research paper thumbnail of Chapter 25 The non-quantal release of acetylcholine from motor nerve terminals: comment on its likely size

Progress in Brain Research, 1993

Research paper thumbnail of Apolipoprotein E4 reduces evoked hippocampal acetylcholine release in adult mice

Journal of neurochemistry, Jan 3, 2015

Apolipoprotein E4 (apoE4) is the most prevalent genetic risk factor for Alzheimer's disease (... more Apolipoprotein E4 (apoE4) is the most prevalent genetic risk factor for Alzheimer's disease (AD). We utilized apoE4-targeted replacement mice (approved by the Tel Aviv University Animal Care Committee) to investigate whether cholinergic dysfunction, which increases during aging and is a hallmark of AD, is accentuated by apoE4. This revealed that levels of the presynaptic cholinergic marker, vesicular acetylcholine transporter in the hippocampus and the corresponding electrically evoked release of acetylcholine, are similar in 4-month-old apoE4 and apoE3 mice. Both parameters decrease with age. This decrease is, however, significantly more pronounced in the apoE4 mice. The levels of cholinacetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) were similar in the hippocampus of young apoE4 and apoE3 mice and decreased during aging. For ChAT this decrease was similar in the apoE4 and apoE3 mice, whereas it was more pronouced in the apoE4 mice, rega...

Research paper thumbnail of Towards predictive docking at aminergic G-protein coupled receptors

Journal of Molecular Modeling, 2015

G protein-coupled receptors (GPCRs) are hard to crystallize. However, attempts to predict their s... more 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. We analyzed the performance of four molecular modeling programs in pose evaluation of re-docked antagonists / inverse agonists to 11 original crystal structures of aminergic GPCRs using an induced fit-docking procedure. AutoDock and Glide were used for docking. AutoDock binding energy function, GlideXP, Prime MM-GB/SA, and YASARA binding function were used for pose scoring. Root mean square deviation (RMSD) of the best pose ranged from 0.09 to 1.58 Å, and median RMSD of the top 60 poses ranged from 1.47 to 3.83 Å. However, RMSD of the top pose ranged from 0.13 to 7.33 Å and ranking of the best pose ranged from the 1st to 60th out of 60 poses. Moreover, analysis of ligand-receptor interactions of top poses revealed substantial differences from interactions found in crystallographic structures. Bad ranking of top poses and discrepancies between top docked poses and crystal structures render current simple docking methods unsuitable for predictive modeling of receptor-ligand interactions. Prime MM-GB/SA optimized for 3NY9 by multiple linear regression did not work well at 3NY8 and 3NYA, structures of the same receptor with different ligands. However, 9 of 11 trajectories of molecular dynamics simulations by Desmond of top poses converged with trajectories of crystal structures. Key interactions were properly detected for all structures. This procedure also worked well for cross-docking of tested β2-adrenergic antagonists. Thus, this procedure represents a possible way to predict interactions of antagonists with aminergic GPCRs.

Research paper thumbnail of Utilization of Superfused Cerebral Slices in Probing Muscarinic Receptor Autoregulation of Acetylcholine Release

Research paper thumbnail of Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms

Pharmacological research : the official journal of the Italian Pharmacological Society, Jan 13, 2015

We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand ... more We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished functional responses to both classical and atypical agonists. O...

Research paper thumbnail of The synthesis and release of acetylcholine in normal and denervated rat diaphragms during incubation in vitro

The Journal of physiology, 1983

1. Normal and denervated rat diaphragms and neural (central) and aneural (peripheral) parts of no... more 1. Normal and denervated rat diaphragms and neural (central) and aneural (peripheral) parts of normal diaphragms were incubated under several different conditions likely to affect the metabolism of acetylcholine (ACh), with the aim of discovering specific features of the control of neural and aneural ACh in the muscle. The concentrations of ACh in the tissue and the medium were measured at the end of the incubations using a radioenzymatic assay, and the amount of ACh synthesized during the incubations was calculated by subtracting the initial amount of ACh present in the tissue from that found in the tissue plus the medium at the end of the incubations.2. Confirming earlier results obtained with bioassays, it was found that, in a medium with 5 mM-K(+) and 2.5 mM-Ca(2+), denervated diaphragms released ACh into the medium at a rate equal to 47% of that observed in normal diaphragms; the amount of ACh released from aneural parts of normal diaphragms was 51% of that released from their ...

Research paper thumbnail of Tacrine (Tetrahydroaminoacridine) and the Metabolism of Acetylcholine and Choline

Phospholipids and Signal Transmission, 1993

Research paper thumbnail of Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor

PLoS ONE, 2010

Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at t... more Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M 1 and M 4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of xanomeline on the binding characteristics and function of the M 1 muscarinic receptor. Pretreatment of CHO cells that stably express the M 1 receptor for 1 hr with increasing concentrations of xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed xanomelineinduced inhibition of [ 3 H]NMS binding from monophasic to biphasic. The high-affinity xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound xanomeline developed with a time course that parallels the return of receptor activation by prebound xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of xanomeline, but not its persistent binding at an allosteric site. Furthermore, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization.

Research paper thumbnail of Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors

PLoS ONE, 2011

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities ... more 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. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G q/11 , while even-numbered receptors prefer coupling to G i/o . We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist highaffinity binding conformation. Antibodies directed against the C-terminus of the a-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [ 3 H]-Nmethylscopolamine. Antibodies directed against the C-terminus of the a-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors precouple with their preferential classes of G-proteins, but only M 1 and M 3 receptors also pre-couple with non-preferential G i/o G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.

Research paper thumbnail of Uncoupling of M1 muscarinic receptor/G-protein interaction by amyloid β1–42

Neuropharmacology, 2013

The overproduction of b-amyloid (Ab) fragments in transgenic APPswe/PS1dE9 mice results in format... more The overproduction of b-amyloid (Ab) 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 transgenedependent weakening of muscarinic receptor-mediated transmission that was not present in young (6e10-week-old) animals but preceded both amyloid deposits and cognitive deficits. Now we investigated immediate and prolonged in vitro effects of non-aggregated Ab 1e42 on coupling of individual muscarinic receptor subtypes expressed in CHO (Chinese hamster ovary) cells and their underlying mechanisms. Immediate application of 1 mM Ab 1e42 had no effect on the binding of the muscarinic antagonist N-methylscopolamine or the agonist carbachol. In contrast, 4-day treatment of CHO cells expressing the M1 muscarinic receptor with 100 nM Ab 1e42 significantly changed the binding characteristics of the muscarinic agonist carbachol and reduced the extent of the M1 receptor-stimulated breakdown of phosphatidylinositol while it did not demonstrate overt toxic effects. The treatment had no influence on the expression of either G-proteins or muscarinic receptors. In concert, we found no change in the gene expression of muscarinic receptor subtypes and gene or protein expression of the G s , G q/11 , and G i/o G-proteins in the cerebral cortex of young adult APPswe/PS1dE9 mice that demonstrate high concentrations of soluble Ab 1e42 and impaired muscarinic receptor-mediated G-protein activation. Our results provide strong evidence that the initial injurious effects of Ab 1e42 on M1 muscarinic receptor-mediated transmissionis is due to compromised coupling of the receptor with G q/11 G-protein. Abbreviations: AD, Alzheimer's disease; CHO cells, Chinese hamster ovary cells; CHO-M1 through CHO-M5, Chinese hamster ovary cells expressing individual subtypes (M1 through M5) of human muscarinic receptors. (H. Janí cková), rudajev@biomed.cas.cz (V. Rudajev), zimcik@biomed.cas.cz (P. Zim cík), jakubik@biomed.cas.cz (J. Jakubík), heikki.tanila@uef.fi (H. Tanila), elfak001@umn.edu (E.E. El-Fakahany), dolezal@ biomed.cas.cz (V. Dole zal).

Research paper thumbnail of The Increase of Choline Acetyltransferase Activity by Docosahexaenoic Acid in NG108-15 Cells Grown in Serum-free Medium is Independent of its Effect on Cell Growth

Neurochemical Research, 2006

We investigated the influence of the polyunsaturated docosahexaenoic acid (22:6n-3; DHA) on the c... more We investigated the influence of the polyunsaturated docosahexaenoic acid (22:6n-3; DHA) on the constitutive expression of choline acetyltransferase (ChAT) in native and induced expression in differentiated cholinergic cells NG108-15 grown in serum-free medium. Elimination of serum-derived trophic support resulted in growth arrest and a strong decrease of ChAT activity. In either conditions, DHA largely rescued general indicators of cell growth and function, and partially prevented the decrease of ChAT activity. However, the maximal effect on general cell state in native and differentiated cells, and ChAT activity in native cells, was reached at or below 10 lmol/l of DHA. In contrast, maximal induction of ChAT activity in differentiated cells required about six times higher concentrations of DHA. These data thus demonstrate stimulatory effect of DHA on ChAT activity that is independent of its general cell protective properties.