Muscarinic Modulation of Synaptic Transmission and Short-Term Plasticity in the Dorsal and Ventral Hippocampus.

Muscarinic neurotransmission is fundamentally involved in supporting several brain functions by modulating flow of information in brain neural circuits including the hippocampus which displays a remarkable functional segregation along its longitudinal axis. However, how muscarinic neuromodulation contributes to the functional segregation along the hippocampus remains unclear. In this study we show that the nonselective muscarinic receptor agonist carbachol similarly suppresses basal synaptic transmission in the dorsal and ventral CA1 hippocampal field, in a concentration-depended manner. Furthermore, using a ten-pulse stimulation train of varying frequency we found that carbachol changes the frequency filtering properties more in ventral than dorsal hippocampus by facilitating synaptic inputs at a wide range of input frequencies in the ventral compared with dorsal hippocampus. Using the M2 receptor antagonist gallamine and the M4 receptor antagonist tropicamide, we found that M2 receptors are involved in controlling basal synaptic transmission and short-term synaptic plasticity (STSP) in the ventral but not the dorsal hippocampus, while M4 receptors participate in modulating basal synaptic transmission and STSP in both segments of the hippocampus. These results were corroborated by the higher protein expression levels of M2 receptors in the ventral compared with dorsal hippocampus. We conclude that muscarinic transmission modulates excitatory synaptic transmission and short-term synaptic plasticity along the entire rat hippocampus by acting through M4 receptors and recruiting M2 receptors only in the ventral hippocampus. Furthermore, M4 receptors appear to exert a permissive role on the actions of M2 receptors on STSP in the ventral hippocampus. This dorsoventral differentiation of muscarinic modulation is expected to have important implications in information processing along the endogenous hippocampal circuitry.

Neuroanatomical Substrates of Circuit-Specific Cholinergic Modulation across the Primate Anterior Cingulate Cortex.

Acetylcholine is a robust neuromodulator of the limbic system and a critical regulator of arousal and emotions. The anterior cingulate cortex (ACC) and the amygdala (AMY) are key limbic structures that are both densely innervated by cholinergic afferents and interact with each other for emotional regulation. The ACC is composed of functionally distinct dorsal (A24), rostral (A32), and ventral (A25) areas that differ in their connections with the AMY. The structural substrates of cholinergic modulation of distinct ACC microcircuits and outputs to AMY are thought to depend on the laminar and subcellular localization of cholinergic receptors. The present study examines the distribution of muscarinic acetylcholine receptors, m1 and m2, on distinct excitatory and inhibitory neurons and on AMY-targeting projection neurons within ACC areas, via immunohistochemistry and injections of neural tracers into the basolateral AMY in adult rhesus monkeys of both sexes. We found that laminar densities of m1+ and m2+ expressing excitatory and inhibitory neurons depended on area and cell type. Among the ACC areas, ventral subgenual ACC A25 exhibited greater m2+ localization on presynaptic inhibitory axon terminals and greater density of m1+ and m2+ expressing AMY-targeting (tracer+) pyramidal neurons. These patterns suggest robust cholinergic disinhibition and potentiation of amygdalar outputs from the limbic ventral ACC, which may be linked to the hyperexcitability of this subgenual ACC area in depression. These findings reveal the anatomical substrate of diverse cholinergic modulation of specific ACC microcircuits and amygdalar outputs that mediate cognitive-emotional integration and dysfunctions underlying stress and affective disorders.

Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

M2 receptors are required for spatiotemporal sequence learning in mouse primary visual cortex.

Acetylcholine is a neurotransmitter that plays a variety of roles in the central nervous system. It was previously shown that blocking muscarinic receptors with a nonselective antagonist prevents a form of experience-dependent plasticity termed "spatiotemporal sequence learning" in the mouse primary visual cortex (V1). Muscarinic signaling is a complex process involving the combined activities of five different G protein-coupled receptors, M1-M5, all of which are expressed in the murine brain but differ from each other functionally and in anatomical localization. Here we present electrophysiological evidence that M2, but not M1, receptors are required for spatiotemporal sequence learning in mouse V1. We show in male mice that M2 is highly expressed in the neuropil in V1, especially in thalamorecipient layer 4, and colocalizes with the soma in a subset of somatostatin-expressing neurons in deep layers. We also show that expression of M2 receptors is higher in the monocular region of V1 than it is in the binocular region but that the amount of experience-dependent sequence potentiation is similar in both regions and that blocking muscarinic signaling after visual stimulation does not prevent plasticity. This work establishes a new functional role for M2-type receptors in processing temporal information and demonstrates that monocular circuits are modified by experience in a manner similar to binocular circuits.NEW & NOTEWORTHY Muscarinic acetylcholine receptors are required for multiple forms of plasticity in the brain and support perceptual functions, but the precise role of the five subtypes (M1-M5) are unclear. Here we show that the M2 receptor is specifically required to encode experience-dependent representations of spatiotemporal relationships in both monocular and binocular regions of mouse V1. This work identifies a novel functional role for M2 receptors in coding temporal information into cortical circuits.

M2 muscarinic receptors negatively modulate cell migration in human glioblastoma cells.

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

Development of a Selective and High Affinity Radioligand, [3H]VU6013720, for the M4 Muscarinic Receptor.

M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.

Long-Term-But Not Short-Term-Plasticity at the Mossy Fiber-CA3 Pyramidal Cell Synapse in Hippocampus Is Altered in M1/M3 Muscarinic Acetylcholine Receptor Double Knockout Mice.

Muscarinic acetylcholine receptors are well-known for their crucial involvement in hippocampus-dependent learning and memory, but the exact roles of the various receptor subtypes (M1-M5) are still not fully understood. Here, we studied how M1 and M3 receptors affect plasticity at the mossy fiber (MF)-CA3 pyramidal cell synapse. In hippocampal slices from M1/M3 receptor double knockout (M1/M3-dKO) mice, the signature short-term plasticity of the MF-CA3 synapse was not significantly affected. However, the rather unique NMDA receptor-independent and presynaptic form of long-term potentiation (LTP) of this synapse was much larger in M1/M3-deficient slices compared to wild-type slices in both field potential and whole-cell recordings. Consistent with its presynaptic origin, induction of MF-LTP strongly enhanced the excitatory drive onto single CA3 pyramidal cells, with the effect being more pronounced in M1/M3-dKO cells. In an earlier study, we found that the deletion of M2 receptors in mice disinhibits MF-LTP in a similar fashion, suggesting that endogenous acetylcholine employs both M1/M3 and M2 receptors to constrain MF-LTP. Importantly, such synergism was not observed for MF long-term depression (LTD). Low-frequency stimulation, which reliably induced LTD of MF synapses in control slices, failed to do so in M1/M3-dKO slices and gave rise to LTP instead. In striking contrast, loss of M2 receptors augmented LTD when compared to control slices. Taken together, our data demonstrate convergence of M1/M3 and M2 receptors on MF-LTP, but functional divergence on MF-LTD, with the net effect resulting in a well-balanced bidirectional plasticity of the MF-CA3 pyramidal cell synapse.

Role of Conserved Tyrosine Lid Residues in the Activation of the M2 Muscarinic Acetylcholine Receptor.

The development of subtype selective small molecule drugs for the muscarinic acetylcholine receptor (mAChR) family has been challenging. The design of more selective ligands can be improved by understanding the structure and function of key amino acid residues that line ligand binding sites. Here we study the role of three conserved key tyrosine residues [Y1043.33, Y4036.51, and Y4267.39 (Ballesteros and Weinstein numbers in superscript)] at the human M2 mAChR, located at the interface between the orthosteric and allosteric binding sites of the receptor. We specifically focused on the role of the three tyrosine hydroxyl groups in the transition between the inactive and active conformations of the receptor by making phenylalanine point mutants. Single-point mutation at either of the three positions was sufficient to reduce the affinity of agonists by ∼100-fold for the M2 mAChR, whereas the affinity of antagonists remained largely unaffected. In contrast, neither of the mutations affected the efficacy of orthosteric agonists. When mutations were combined into double and triple M2 mAChR mutants, the affinity of antagonists was reduced by more than 100-fold compared with the wild-type M2 receptor. In contrast, the affinity of allosteric modulators, either negative or positive, was retained at all single and multiple mutations, but the degree of allosteric effect exerted on the endogenous ligand acetylcholine was affected at all mutants containing Y4267.39F. These findings will provide insights to consider when designing future mAChR ligands. SIGNIFICANCE STATEMENT: Structural studies demonstrated that three tyrosine residues between the orthosteric and allosteric sites of the M2 muscarinic acetylcholine receptor (mAChR) had different hydrogen bonding networks in the inactive and active conformations. The role of hydroxyl groups of the tyrosine residues on orthosteric and allosteric ligand pharmacology was unknown. We found that hydroxyl groups of the tyrosine residues differentially affected the molecular pharmacology of orthosteric and allosteric ligands. These results provide insights to consider when designing future mAChR ligands.

Application of Mixed-Solvent Molecular Dynamics Simulations for Prediction of Allosteric Sites on G Protein-Coupled Receptors.

The development of small molecule allosteric modulators acting at G protein-coupled receptors (GPCRs) is becoming increasingly attractive. Such compounds have advantages over traditional drugs acting at orthosteric sites on these receptors, in particular target specificity. However, the number and locations of druggable allosteric sites within most clinically relevant GPCRs are unknown. In the present study, we describe the development and application of a mixed-solvent molecular dynamics (MixMD)-based method for the identification of allosteric sites on GPCRs. The method employs small organic probes with druglike qualities to identify druggable hotspots in multiple replicate short-timescale simulations. As proof of principle, we first applied the method retrospectively to a test set of five GPCRs (cannabinoid receptor type 1, C-C chemokine receptor type 2, M2 muscarinic receptor, P2Y purinoceptor 1, and protease-activated receptor 2) with known allosteric sites in diverse locations. This resulted in the identification of the known allosteric sites on these receptors. We then applied the method to the µ-opioid receptor. Several allosteric modulators for this receptor are known, although the binding sites for these modulators are not known. The MixMD-based method revealed several potential allosteric sites on the mu-opioid receptor. Implementation of the MixMD-based method should aid future efforts in the structure-based drug design of drugs targeting allosteric sites on GPCRs. SIGNIFICANCE STATEMENT: Allosteric modulation of G protein-coupled receptors (GPCRs) has the potential to provide more selective drugs. However, there are limited structures of GPCRs bound to allosteric modulators, and obtaining such structures is problematic. Current computational methods utilize static structures and therefore may not identify hidden or cryptic sites. Here we describe the use of small organic probes and molecular dynamics to identify druggable allosteric hotspots on GPCRs. The results reinforce the importance of protein dynamics in allosteric site identification.

Clinical and prognostic associations of autoantibodies recognizing adrenergic/muscarinic receptors in patients with heart failure.

AIMS: The importance of autoantibodies (AABs) against adrenergic/muscarinic receptors in heart failure (HF) is not well-understood. We investigated the prevalence and clinical/prognostic associations of four AABs recognizing the M2-muscarinic receptor or the ß1-, ß2-, or ß3-adrenergic receptor in a large and well-characterized cohort of patients with HF. METHODS AND RESULTS: Serum samples from 2256 patients with HF from the BIOSTAT-CHF cohort and 299 healthy controls were analysed using newly established chemiluminescence immunoassays. The primary outcome was a composite of all-cause mortality and HF rehospitalization at 2-year follow-up, and each outcome was also separately investigated. Collectively, 382 (16.9%) patients and 37 (12.4%) controls were seropositive for ≥1 AAB (P = 0.045). Seropositivity occurred more frequently only for anti-M2 AABs (P = 0.025). Amongst patients with HF, seropositivity was associated with the presence of comorbidities (renal disease, chronic obstructive pulmonary disease, stroke, and atrial fibrillation) and with medication use. Only anti-ß1 AAB seropositivity was associated with the primary outcome [hazard ratio (95% confidence interval): 1.37 (1.04-1.81), P = 0.024] and HF rehospitalization [1.57 (1.13-2.19), P = 0.010] in univariable analyses but remained associated only with HF rehospitalization after multivariable adjustment for the BIOSTAT-CHF risk model [1.47 (1.05-2.07), P = 0.030]. Principal component analyses showed considerable overlap in B-lymphocyte activity between seropositive and seronegative patients, based on 31 circulating biomarkers related to B-lymphocyte function. CONCLUSIONS: AAB seropositivity was not strongly associated with adverse outcomes in HF and was mostly related to the presence of comorbidities and medication use. Only anti-ß1 AABs were independently associated with HF rehospitalization. The exact clinical value of AABs remains to be elucidated.

Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor.

The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and ß-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance ß-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.

Associations of common genetic risk variants of the muscarinic acetylcholine receptor M2 with cardiac autonomic dysfunction in patients with schizophrenia.

OBJECTIVES: Decreased vagal modulation, which has consistently been observed in schizophrenic patients, might contribute to increased cardiac mortality in schizophrenia. Previously, associations between CHRM2 (Cholinergic Receptor Muscarinic 2) and cardiac autonomic features have been reported. Here, we tested for possible associations between these polymorphisms and heart rate variability in patients with schizophrenia. METHODS: A total of three single nucleotide polymorphisms (SNPs) in CHRM2 (rs73158705 A>G, rs8191992 T>A and rs2350782 T>C) that achieved significance (p < 5 * 10-8) in genome-wide association studies for cardiac autonomic features were genotyped in 88 drug-naïve patients, 61 patients receiving antipsychotic medication and 144 healthy controls. Genotypes were analysed for associations with parameters of heart rate variability and complexity, in each diagnostic group. RESULTS: We observed a significantly altered heart rate variability in unmedicated patients with identified genetic risk status in rs73158705 A>G, rs8191992 T>A and rs2350782 T>C as compared to genotype non-risk status. In patients receiving antipsychotic medication and healthy controls, these associations were not observed. DISCUSSION: We report novel candidate genetic associations with cardiac autonomic dysfunction in schizophrenia, but larger cohorts are required for replication.

Determination of Region-Specific Roles of the M3 Muscarinic Acetylcholine Receptor in Gastrointestinal Motility.

BACKGROUND: The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. AIMS: The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. METHODS: We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. RESULTS: PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127 µM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30 mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. CONCLUSIONS: This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.

M2 Muscarinic Receptor-Dependent Contractions of Airway Smooth Muscle are Inhibited by Activation of ß-Adrenoceptors.

Beta-adrenoceptor (ß-AR) agonists inhibit cholinergic contractions of airway smooth muscle (ASM), but the underlying mechanisms are unclear. ASM cells express M3 and M2 muscarinic receptors, but the bronchoconstrictor effects of acetylcholine are believed to result from activation of M3Rs, while the role of the M2Rs is confined to offsetting ß-AR-dependent relaxations. However, a profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM was recently reported, indicating an important role for M2Rs in cholinergic contractions of ASM. Here, we investigated if M2R-dependent contractions of murine bronchial rings were inhibited by activation of ß-ARs. M2R-dependent contractions were apparent at low frequency (2Hz) electric field stimulation (EFS) and short (10s) stimulus intervals. The ß1-AR agonist, denopamine inhibited EFS-evoked contractions of ASM induced by reduction in stimulus interval from 100 to 10 s and was more effective at inhibiting contractions evoked by EFS at 2 than 20 Hz. Denopamine also abolished carbachol-evoked contractions that were resistant to the M3R antagonist 4-DAMP, similar to the effects of the M2R antagonists, methoctramine and AFDX-116. The inhibitory effects of denopamine on EFS-evoked contractions of ASM were smaller in preparations taken from M2R -/- mice, compared to wild-type (WT) controls. In contrast, inhibitory effects of the ß3-AR agonist, BRL37344, on EFS-evoked contractions of detrusor strips taken from M2R -/- mice were greater than WT controls. These data suggest that M2R-dependent contractions of ASM were inhibited by activation of ß1-ARs and that genetic ablation of M2Rs decreased the efficacy of ß-AR agonists on cholinergic contractions.

Click Chemistry-Enabled Conjugation Strategy for Producing Dibenzodiazepinone-Type Fluorescent Probes To Target M2 Acetylcholine Receptors.

The development of fluorescently labeled receptor-targeting compounds represents a powerful pharmacological tool to study and characterize ligand-receptor interactions. Despite significant advances in developing sub-type-specific antagonists for muscarinic acetylcholine receptors (mAChRs), reports on antagonists feasible for click chemistry are less common. Here, we designed and synthesized an antagonist suitable for probe attachment through click chemistry, namely, dibenzodiazepinone (DIBA)-alkyne, based on a previously reported DIBA scaffold with a high binding affinity to type-2 mAChR (M2R). To demonstrate the versatility of DIBA-alkyne as a building block for bioconjugates, we assembled DIBA-alkyne with Cyanine5 fluorophores (Cy5) and polyethylene glycol (PEG) biomolecules to obtain fluorescent DIBA antagonist (DIBA-Cy5) and fluorescent DIBA PEG derivatives. Flow cytometric analysis showed that DIBA-Cy5 possessed a high binding affinity to M2R (Kd = 1.80 nM), a two-order magnitude higher binding affinity than M1R. Fluorescent DIBA PEG derivatives maintained a potent binding to the M2R (Kd ≤ 4 nM), confirmed by confocal microscopic imaging. Additionally, DIBA-Cy5 can serve as a fluorescent ligand in the receptor-ligand competitive binding assay for other mAChR ligands, an attractive alternative to the traditional radioligand-based assay. The competitive binding mode between DIBA-Cy5 and orthosteric antagonist atropine/allosteric modulator LY2119620 indicated a dualsteric binding mode of the DIBA-type antagonist to M2R. Lastly, we demonstrated the direct staining of DIBA-Cy5 to M2R receptors in the sinoatrial node of a mouse heart. The adaptability of the clickable DIBA antagonist to a wide range of fluorophores and biomolecules can facilitate its use in various biomedical applications such as binding assays that screen compounds for M2R as the receptor target.

Functional antibodies against G-protein coupled receptors in Beagle dogs infected with two different strains of Trypanosoma cruzi.

The interaction of the anti-beta1-adrenergic receptor autoantibodies (ß1ARAb) and the anti-muscarinic M2 receptor autoantibodies (M2RAb) with cardiac neurotransmitter receptors were identified in human chronic Chagas cardiomyopathy (CCC) related to the ECG and dysautonomia disturbances. Dogs are considered gold model to the study of Trypanosoma cruzi infection due the clinical similarities with CCC. This study aims to evaluate whether anti-ß1ARAb, anti-ß2ARAb, and anti-muscarinic M2RAb are generated in Beagle dogs infected by T. cruzi using Y and Berenice-78 strains of T. cruzi. Animals were infected with 4.0 x 103 bloodstream trypomastigotes/kg of body weight and, after 25 months of infection, blood sample was collected, and serum stored at -80°C. Dog serum was treated by ammonium sulphate precipitation and the IgG antibodies isolated and added to the beating neonatal rats' cardiomyocytes. All T. cruzi-infected dogs developed agonistic ß1ARAb, ß2ARAb, and M2RAb. Animals infected by Berenice strain presented less ß2ARAb and M2RAb activities than dogs infected by Y strain of the parasite. In cardiomyocytes culture, the antibodies recognized an epitope on the second extracellular loop of the receptors which were similar to findings in human Chagas disease. There was no detection of antibody against G protein-coupled receptor in serum from uninfected dogs. In conclusion, both Y and Berenice-78 strains of T. cruzi induced dog antibodies, whose targets located in the second extracellular loop of the adrenergic and muscarinic receptors were similar to those observed in individuals with CCC. Therefore, our findings highlight dogs as a promisor model to investigate pathogenic roles of functional Ab against G-protein coupled receptors.

Neural Subtype-dependent Cholinergic Modulation of Neural Activities by Activation of Muscarinic 2 Receptors and G Protein-activated Inwardly Rectifying Potassium Channel in Rat Periaqueductal Gray Neurons.

Acetylcholine plays a pivotal role in the regulation of functions such as pain and the sleep and wake cycle by modulating neural activities of the ventrolateral periaqueductal gray (vlPAG). Electrophysiological studies have shown that cholinergic effects are inconsistent among recorded neurons, particularly in the depolarization and hyperpolarization of the resting membrane potential (RMP). This discrepancy may be due to the neural subtype-dependent cholinergic modulation of the RMP. To examine this possibility, we performed whole-cell patch-clamp recordings from subtype-identified neurons using vesicular GABA transporter (VGAT)-Venus × ChAT-TdTomato rats and elucidated cellular mechanisms of cholinergic effects on the RMP. The application of carbachol hyperpolarized the RMP of cholinergic neurons in a dose-dependent manner but had much less of an effect on other neural subtypes, including GABAergic/glycinergic and glutamatergic neurons. Cholinergic hyperpolarization was accompanied by a decrease in input resistance. These cholinergic effects were blocked by AF-DX384 or gallamine and were mimicked by arecaidine but-2-ynyl ester tosylate, suggesting that the carbachol-induced hyperpolarization of the RMP in cholinergic neurons is mediated via M2 receptors. Tertiapin suppressed the carbachol-induced G protein-activated inwardly rectifying potassium channel (GIRK) currents and hyperpolarization of the RMP in cholinergic neurons. Intracellular application of GDP-ß-S blocked the carbachol-induced hyperpolarization of the RMP. Neostigmine slowly hyperpolarized the RMP in cholinergic neurons. These results suggest that neural firing of vlPAG cholinergic neurons is suppressed by GIRK currents induced via M2 receptor activation, and this negative feedback regulation of cholinergic neuronal activities can be induced by acetylcholine, which is intrinsically released in the vlPAG.

The activation of M2 muscarinic receptor inhibits cell growth and survival in human epithelial ovarian carcinoma.

Ovarian cancer is the fifth leading cause of cancer-related deaths in females. Many ovarian tumor cell lines express muscarinic receptors (mAChRs), and their expression is correlated with reduced survival of patients. We have characterized the expression of mAChRs in two human ovarian carcinoma cell lines (SKOV-3, TOV-21G) and two immortalized ovarian surface epithelium cell lines (iOSE-120, iOSE-398). Among the five subtypes of mAChRs (M1-M5 receptors), we focused our attention on the M2 receptor, which is involved in the inhibition of tumor cell proliferation. Western blot analysis and real-time PCR analyses indicated that the levels of M2 are statistically downregulated in cancer cells. Therefore, we investigated the effect of arecaidine propargyl ester hydrobromide (APE), a preferential M2 agonist, on cell growth and survival. APE treatment decreased cell number in a dose and time-dependent manner by decreasing cell proliferation and increasing cell death. FACS and immunocytochemistry analysis have also demonstrated the ability of APE to accumulate the cells in G2/M phase of the cell cycle and to increase the percentage of abnormal mitosis. The higher level of M2 receptors in the iOSE cells rendered these cells more sensitive to APE treatment than cancer cells. The data here reported suggest that M2 has a negative role in cell growth/survival of ovarian cell lines, and its downregulation may favor tumor progression.