105 research outputs found
FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists
Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in FΓΆrster resonance energy transfer (FRET).Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M(1) mAChR, respectively. The optimized FRET receptor construct (M(1)-cam5) was expressed stably in HEK293 cells.The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with GΞ±(q/11) proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M(1) FRET (F(EYFP)/F(ECFP)) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.The M(1) FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M(1) mAChR activation
Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs
Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimerβs disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including βepileptic-likeβ seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD
Steady-state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B
Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a Ξ²-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-Ξ²-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone
Clustered Coding Variants in the Glutamate Receptor Complexes of Individuals with Schizophrenia and Bipolar Disorder
Current models of schizophrenia and bipolar disorder implicate multiple genes,
however their biological relationships remain elusive. To test the genetic role
of glutamate receptors and their interacting scaffold proteins, the exons of ten
glutamatergic βhubβ genes in 1304 individuals were re-sequenced in
case and control samples. No significant difference in the overall number of
non-synonymous single nucleotide polymorphisms (nsSNPs) was observed between
cases and controls. However, cluster analysis of nsSNPs identified two exons
encoding the cysteine-rich domain and first transmembrane helix of GRM1 as a
risk locus with five mutations highly enriched within these domains. A new
splice variant lacking the transmembrane GPCR domain of GRM1 was discovered in
the human brain and the GRM1 mutation cluster could perturb the regulation of
this variant. The predicted effect on individuals harbouring multiple mutations
distributed in their ten hub genes was also examined. Diseased individuals
possessed an increased load of deleteriousness from multiple concurrent rare and
common coding variants. Together, these data suggest a disease model in which
the interplay of compound genetic coding variants, distributed among glutamate
receptors and their interacting proteins, contribute to the pathogenesis of
schizophrenia and bipolar disorders
Alternative splicing of G protein-coupled receptors: physiology and pathophysiology
The G protein-coupled receptors (GPCRs) are a superfamily of transmembrane receptors that have a broad distribution and can collectively recognise a diverse array of ligands. Activation or inhibition of GPCR signalling can affect many (patho)physiological processes, and consequently they are a major target for existing and emerging drug therapies. A common observation has been that the pharmacological, signalling and regulatory properties of GPCRs can differ in a cell- and tissue-specific manner. Such βphenotypicβ diversity might be attributable to post-translational modifications and/or association of GPCRs with accessory proteins, however, post-transcriptional mechanisms are also likely to contribute. Although approximately 50% of GPCR genes are intronless, those that possess introns can undergo alternative splicing, generating GPCR subtype isoforms that may differ in their pharmacological, signalling and regulatory properties. In this review we shall highlight recent research into GPCR splice variation and discuss the potential consequences this might have for GPCR function in health and disease
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