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. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to Gq/11, while even-numbered receptors prefer coupling to Gi/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 high-affinity binding conformation. Antibodies directed against the C-terminus of the α-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 [3H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-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 pre-couple with their preferential classes of G-proteins, but only M1 and M3 receptors also pre-couple with non-preferential Gi/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

Functional dissociation of behavioral effects from acetylcholine and glutamate released from cholinergic striatal interneurons

In the striatum, cholinergic interneurons (CINs) have the ability to release both acetylcholine and glutamate, due to the expression of the vesicular acetylcholine transporter (VAChT) and the vesicular glutamate transporter 3 (VGLUT3). However, the relationship these neurotransmitters have in the regulation of behavior is not fully understood. Here we used reward-based touchscreen tests in mice to assess the individual and combined contributions of acetylcholine/glutamate co-transmission in behavior. We found that reduced levels of the VAChT from CINs negatively impacted dopamine signalling in response to reward, and disrupted complex responses in a sequential chain of events. In contrast, diminished VGLUT3 levels had somewhat opposite effects. When mutant mice were treated with haloperidol in a cue-based task, the drug did not affect the performance of VAChT mutant mice, whereas VGLUT3 mutant mice were highly sensitive to haloperidol. In mice where both vesicular transporters were deleted from CINs, we observed altered reward-evoked dopaminergic signalling and behavioral deficits that resemble, but were worse, than those in mice with specific loss of VAChT alone. These results demonstrate that the ability to secrete two different neurotransmitters allows CINs to exert complex modulation of a wide range of behaviors

Cholinergic transmission on muscarinic receptors: the influence of β-amyloid and the role of individual G proteins

Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

Muscarinic acetylcholine transmission and Alzheimer's disease

Souhrn Poškození cholinergního neurotransmiterového systému se pravidelně objevuje u zvířecích modelů Alzheimerovy choroby stejně tak jako v mozku pacientů s tímto onemocněním. Kromě toho některé výsledky ukazují, že aktivace jednotlivých podtypů mAChR specificky ovlivňuje štěpení APP, které může probíhat amyloidogenním či neamyloidogenním způsobem a určuje tak množství vytvořeného neurotoxického β-amyloidu. Ve své práci jsem studovala vliv akutního a dlouhodobého působení Aβ1-42 na mAChR, které tvoří významnou součást cholinergního systému. Z výsledků mé práce vyplývá, že Aβ1-42 dlouhodobě přítomný v kultivačním médiu s rostoucími buňkami exprimujícími jednotlivé podtypy mAChR negativně ovlivňuje vazebné i funkční vlastnosti některých lichých podtypů mAChR (především M1, méně M3). Podobné poškození funkce mAChR jsem zaznamenala také v mozkové kůře transgenních myší APPswe/PS1dE9, které jsou jedním z běžně používaných zvířecích modelů Alzheimerovy choroby. Postižení muskarinové transmise se u myšího modelu Alzheimerovy choroby objevuje během postupného zvyšování koncentrace rozpustného β-amyloidu (zejména fragmentu Aβ1-42), tj. dříve než typická patologie nebo behaviorální poruchy, a lze ho napodobit in vitro působením nízké koncentrace Aβ1-42. Tato pozorování podporují představu časného postižení...Impairment of the cholinergic neurotransmission system is regularly detected in animal models of Alzheimer's disease as well as in human patients suffering from this serious disease. Moreover, there is increasing amount of evidence suggesting that activation of individual mAChR subtypes specifically influences the cleavage of APP, the precursor for β-amyloid. APP can be processed in an amyloidogenic or non-amyloidogenic pathway and a relative abundance of these patways contributes to establishing the final concentration of neurotoxic β-amyloid in the brain. In this work, I have studied the acute and chronic effects of A β1-42 on binding and functional characteristics of mAChR. I have demonstrated that Aβ1-42 present in cell culture expressing the individual subtypes of mAChR negatively and specifically influences the function of the M1 mAChR subtype. I have also detected a decline in muscarinic receptor-mediated signal transduction in brain tissue of young adult APPswe/PS1dE9 mice, a commonly used animal model of Alzheimer's disease. Demonstration of the impairment of muscarinic transmissin in transgenic mice by soluble β-amyloid that occurs earlier than amyloid pathology and behavioral deficit, and its imitation by soluble Aβ1-42 in vitro lend strong support to the notion of the early involvement...Department of Neurology First Faculty of Medicine and General University Hospital in PragueNeurologická klinika 1. LF UK a VFN v PrazeFirst Faculty of Medicine1. lékařská fakult

Muscarinic acetylcholine transmission and Alzheimer's disease

Impairment of the cholinergic neurotransmission system is regularly detected in animal models of Alzheimer's disease as well as in human patients suffering from this serious disease. Moreover, there is increasing amount of evidence suggesting that activation of individual mAChR subtypes specifically influences the cleavage of APP, the precursor for β-amyloid. APP can be processed in an amyloidogenic or non-amyloidogenic pathway and a relative abundance of these patways contributes to establishing the final concentration of neurotoxic β-amyloid in the brain. In this work, I have studied the acute and chronic effects of A β1-42 on binding and functional characteristics of mAChR. I have demonstrated that Aβ1-42 present in cell culture expressing the individual subtypes of mAChR negatively and specifically influences the function of the M1 mAChR subtype. I have also detected a decline in muscarinic receptor-mediated signal transduction in brain tissue of young adult APPswe/PS1dE9 mice, a commonly used animal model of Alzheimer's disease. Demonstration of the impairment of muscarinic transmissin in transgenic mice by soluble β-amyloid that occurs earlier than amyloid pathology and behavioral deficit, and its imitation by soluble Aβ1-42 in vitro lend strong support to the notion of the early involvement..

Rates of basal and carbachol-stimulated association of [³⁵S]GTPγS in CHO membranes expressing wild type and mutant M₁ receptors.

<p>Data are means ± S.E.M. From 3 experiments performed in quadruplicates.</p><p>*, Significantly different from control;</p>#<p>, significantly different from wild type (M₁ wt), t-test (P<0.05).</p

Effects of anti-G-protein antibodies on competition between different agonists and [³H]NMS binding at M₂ receptors.

<p>Binding of 1 nM [³H]NMS to membranes from CHO cells expressing M₂ receptors in the presence of increasing concentrations (abscissa, log M) of the agonists carbachol (upper left), furmethide (upper right), oxotremorine (lower left) and pilocarpine (lower right) is expressed as per cent of control binding in the absence of agonist. Filled circles, control binding in the absence of antibodies. Open symbols, binding in the presence of anti-G_i/o (circles), anti-G_s/olf (squares) and anti-G_q/11 (diamonds) antibodies. Data are means ± S.E.M of values from 3 experiments performed in quadruplicates. Curves were fitted using equation 3 and results of fits are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027732#pone-0027732-t004" target="_blank">Table 4</a>.</p

Effects of anti-G-protein antibodies on competition between carbachol and [³H]NMS binding at M₁ to M₄ receptors.

<p>Binding of 1 nM [³H]NMS to membranes from CHO cells expressing M₁ (upper left), M₂ (upper right), M₃ (lower left) and M₄ (lower right) receptors in the presence of increasing concentrations (abscissa, log M) of carbachol is expressed as per cent of control binding in the absence of carbachol. Filled circles, control binding in the absence of antibodies. Open symbols, binding in the presence of anti-G_i/o (circles), anti-G_s/olf (squares), and anti-G_q/11 (diamonds) antibodies. Data are means ± S.E.M of values from 3 experiments performed in quadruplicates. Curves were fitted using equation 3 and results of fits are shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027732#pone-0027732-t002" target="_blank">Table 2</a>.</p

Effects of anti-G-protein antibodies on competition between agonists and [³H]NMS binding at M₂ receptors after suppression of G_i/o G-proteins expression.

<p>Binding of 1 nM [³H]NMS to membranes from CHO cells expressing M₂ receptors after suppression of expression of G_i/o G-proteins by siRNA was determined in the presence of increasing concentrations (abscissa, log M) of the agonists carbachol (upper left), furmethide (upper right), oxotremorine (lower left), and pilocarpine (lower right). Binding is expressed as per cent of control binding in the absence of agonist. Filled circles, control binding in the absence of antibodies. Open symbols, binding in the presence of anti-G_i/o (circles), anti-G_s/olf (squares) and anti-G_q/11 (diamonds) antibodies. Data are means ± S.E.M of values from 3 experiments performed in quadruplicates. Curves were fitted using equation 3 and results of fits are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027732#pone-0027732-t006" target="_blank">Table 6</a>.</p

Stimulation of [³⁵S]GTPγS binding by carbachol at the M₂ receptor after suppression of expression of G_i/o G-proteins.

<p>M₂ receptor-mediated stimulation of [³⁵S]GTPγS binding to G_i/o (circles), G_s/olf (squares) and G_q/11 G-proteins (diamonds) after suppression of expression of G_i/o G-proteins by siRNA was stimulated by increasing concentrations of carbachol (abscissa, log M). Response is expressed as fold over basal (ordinate). Data are means ± S.E.M of values from 3 experiments performed in quadruplicates. Curves were fitted using equation 2 and results of fits are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027732#pone-0027732-t005" target="_blank">Table 5</a>.</p