Muscarinic Toxin Research Articles

Antagonism of the Muscarinic Acetylcholine Type 1 Receptor Enhances Mitochondrial Membrane Potential and Expression of Respiratory Chain Components via AMPK in Human Neuroblastoma SH-SY5Y Cells and Primary Neurons

Impairments in mitochondrial physiology play a role in the progression of multiple neurodegenerative conditions, including peripheral neuropathy in diabetes. Blockade of muscarinic acetylcholine type 1 receptor (M1R) with specific/selective antagonists prevented mitochondrial dysfunction and reversed nerve degeneration in in vitro and in vivo models of peripheral neuropathy. Specifically, in type 1 and type 2 models of diabetes, inhibition of M1R using pirenzepine or muscarinic toxin 7 (MT7) induced AMP-activated protein kinase (AMPK) activity in dorsal root ganglia (DRG) and prevented sensory abnormalities and distal nerve fiber loss. The human neuroblastoma SH-SY5Y cell line has been extensively used as an in vitro model system to study mechanisms of neurodegeneration in DRG neurons and other neuronal sub-types. Here, we tested the hypothesis that pirenzepine or MT7 enhance AMPK activity and via this pathway augment mitochondrial function in SH-SY5Y cells. M1R expression was confirmed by utilizing a fluorescent dye, ATTO590-labeled MT7, that exhibits great specificity for this receptor. M1R antagonist treatment in SH-SY5Y culture increased AMPK phosphorylation and mitochondrial protein expression (OXPHOS). Mitochondrial membrane potential (MMP) was augmented in pirenzepine and MT7 treated cultured SH-SY5Y cells and DRG neurons. Compound C or AMPK-specific siRNA suppressed pirenzepine or MT7-induced elevation of OXPHOS expression and MMP. Moreover, muscarinic antagonists induced hyperpolarization by activating the M-current and, thus, suppressed neuronal excitability. These results reveal that negative regulation of this M1R-dependent pathway could represent a potential therapeutic target to elevate AMPK activity, enhance mitochondrial function, suppress neuropathic pain, and enhance nerve repair in peripheral neuropathy.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

Selective or Specific M1 Muscarinic Receptor Antagonists Exhibit Biased Agonism at the M1 Receptor via β‐arrestin Signaling

Introduction Selective (pirenzepine; PZ) and specific (muscarinic toxin 7; MT7) antagonists of the muscarinic acetylcholine type 1 receptor (M1R) reverse nerve degeneration in rodent models of peripheral neuropathy. These drugs drive axonal outgrowth of adult sensory neurons and signaling is mediated via extracellular-regulated protein kinase (ERK) and β-arrestin. In order to understand the pharmacological mechanism of action of PZ and MT7, we hypothesized that these drugs exhibited biased agonism at the M1R mediated via b-arrestin signaling. Methods Nano bioluminescence resonance energy transfer (NanoBRET) assay: Human embryonic kidney (HEK293) cells co-expressing human M1R-Nluc and Halo-tagged β-arrestin 2 were treated with different doses of M1R agonists (muscarine or carbachol) or antagonists (PZ and MT7)at different time points. NanoBRET assay was employed to measure ligand-induced β-arrestin 2 recruitment to the M1R. Assays were performed in triplicate with 3 independent experiments completed. Data are presented as corrected milli-BRET (mBRET) units. Substrate was added 2 min before BRET detection, and drugs were applied at various time points. Data are reported as the mean ± SD (statistics by 1-way ANOVA with Tukey's post-hoc test). Inositol-phosphate one (IP1) measurement: Intracellular IP1 levels were measured using IP-One assay (Cisbio Bioassays, USA). HEK293 cells co-expressing hM1R-Nluc and Halo-tagged β-arrestin 2 were plated onto 384-well white microplates (20000 cells per well) and were treated with stimulation buffer with or without 1) different doses of M1R agonist (muscarine), 2) M1R antagonists (PZ and MT7) or 3) both M1R antagonist (different doses of PZ) and agonist (muscarine, 1mM) for different time points at 37 °C. Cells were lysed by addition of the supplied buffer containing d2-labeled IP1, followed by addition of terbium cryptate-labeled anti-IP1 antibody, according to the manufacturer's instructions. Plates were incubated for 1 h at room temperature and fluorescence signals were measured at 620 and 665 nm using the Biotek Synergy Neo2 plate reader. HTRF ratio 665 nm/620 nm of each well were extrapolated on the standard curve to obtain IP1 concentrations. Results Both carbachol (Fig. 1A) and muscarine (Fig. 1B) induced Halo-tagged β-arrestin 2 recruitment to hM1R-Nluc in a dose-dependent manner at 5 min of treatment. pirenzepine (Fig. 1C) and MT7 (Fig. 1D) induced Halo-tagged β-arrestin 2 recruitment to hM1R-Nluc in a dose-dependent manner at longer time points (e.g. after 30 min). Interestingly, over short periods, e.g. 2-3 min, PZ and MT7 acted as classical antagonists with blockade of muscarine-induced b-arrestin 2 association with M1R. Unlike MT7 and PZ, muscarine increased IP1 levels in transfected cells (Fig. 2A). Also, PZ dose-dependently inhibited the formation of IP1 following muscarine (1mM) treatment (Fig. 2B). Conclusion Thisstudy for the first time provides molecular evidence to support a role for selective/specific muscarinic receptor antagonists acting as biased agonists at the M1R. This novel signaling pathway may contribute to driving axonal regeneration in adult sensory neurons and could be mobilized for nerve repair in neuropathic disease.

Structure and selectivity engineering of the M 1 muscarinic receptor toxin complex

Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M1AChR in complex with MT7, a subtype-selective anti-M1AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M1AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M1AChR toward M2AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein-coupled receptor modulators.

Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase \u03b2 to Augment Mitochondrial Function and Prevent Neurodegeneration

Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M1R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKβ to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M1R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 μM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 μM) or short hairpin RNA to CaMKKβ. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 μM). PGC-1α reporter activity was augmented up to 2-fold (p < 0.05) by MT7 and blocked by STO-609. Mitochondrial maximal respiration and spare respiratory capacity were elevated after 3 h of exposure to MT7 (p < 0.05). Diabetes and CIPN induced a significant (p < 0.05) decrease in corneal nerve density which was corrected by topical delivery of MT7. We reveal a novel M1R-modulated, CaMKKβ-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.

Augmentation of Endogenous Acetylcholine Uptake and Cholinergic Facilitation of Hippocampal Long-Term Potentiation by Acetylcholinesterase Inhibition

Hippocampal cholinergic activity enhances long-term potentiation (LTP) of synaptic transmission in intrahippocampal circuits and regulates cognitive function. We recently demonstrated intracellular distribution of functional M1-muscarinic acetylcholine receptors (mAChRs) and neuronal uptake of acetylcholine (ACh) in the central nervous system. Here we examined whether endogenous ACh acts on intracellular M1-mAChRs following its uptake and causes cholinergic facilitation of hippocampal LTP. ACh esterase (AChE) activities and [3H]ACh uptake was measured in rat hippocampal segments. LTP of evoked field excitatory postsynaptic potentials at CA1 synapses was induced by high frequency stimulation in hippocampal slices. Pretreatment with diisopropylfluorophosphate (DFP) irreversibly inhibited AChE, augmented ACh uptake, and significantly enhanced the LTP. This cholinergic facilitation was inhibited by pirenzepine, a membrane-permeable M1 antagonist, while only the early stage of cholinergic facilitation was inhibited by a membrane-impermeable M1 antagonist, muscarinic toxin 7. Tetraethylammonium (TEA) inhibited ACh uptake in hippocampal segments and selectively suppressed late stage cholinergic facilitation without changing the early stage. In contrast, LTP in DFP-untreated slices was not affected by the muscarinic antagonists and TEA. Carbachol (CCh; an AChE-resistant muscarinic agonist) competed with ACh for its uptake and produced cholinergic facilitation of LTP in DFP-untreated slices. The late stage of CCh-induced facilitation was also selectively inhibited by TEA. Our results suggest that when AChE is inactivated by inhibitors, LTP in hippocampal slices is significantly enhanced by endogenous ACh and that cholinergic facilitation is caused by direct activation of cell-surface M1-mAChRs and subsequent activation of intracellular M1-mAChRs after ACh uptake.

Last decade update for three-finger toxins: Newly emerging structures and biological activities.

Three-finger toxins (TFTs) comprise one of largest families of snake venom toxins. While they are principal to and the most toxic components of the venoms of the Elapidae snake family, their presence has also been detected in the venoms of snakes from other families. The first TFT, α-bungarotoxin, was discovered almost 50 years ago and has since been used widely as a specific marker of the α7 and muscle-type nicotinic acetylcholine receptors. To date, the number of TFT amino acid sequences deposited in the UniProt Knowledgebase free-access database is more than 700, and new members are being added constantly. Although structural variations among the TFTs are not numerous, several new structures have been discovered recently; these include the disulfide-bound dimers of TFTs and toxins with nonstandard pairing of disulfide bonds. New types of biological activities have also been demonstrated for the well-known TFTs, and research on this topic has become a hot topic of TFT studies. The classic TFTs α-bungarotoxin and α-cobratoxin, for example, have now been shown to inhibit ionotropic receptors of γ-aminobutyric acid, and some muscarinic toxins have been shown to interact with adrenoceptors. New, unexpected activities have been demonstrated for some TFTs as well, such as toxin interaction with interleukin or insulin receptors and even TFT-activated motility of sperm. This minireview provides a summarization of the data that has emerged in the last decade on the TFTs and their activities.

Differences among muscarinic agonists in M1 receptor-mediated nonselective cation channel activation and TASK1 channel inhibition in adrenal medullary cells

Muscarinic receptor stimulation induces depolarizing inward currents and catecholamine secretion in adrenal medullary (AM) cells from various mammals. In guinea-pig AM cells muscarine and oxotremorine at concentrations ≤ 1 μM produce activation of nonselective cation channels with a similar potency and efficacy, whereas muscarine at higher concentrations produces not only nonselective cation channel activation, but also TASK1 channel inhibition. In rat AM cells, the muscarinic M1 receptor is involved in TASK1 channel inhibition in response to muscarinic agonists, and the efficacy of oxotremorine is half that of muscarine. These pharmacological findings might indicate that different muscarinic receptor subtypes are responsible for the regulation of nonselective cation and TASK1 channel activities. The present study aimed to determine the muscarinic receptor subtypes involved in nonselective cation channel activation in guinea-pig and mouse AM cells. The inward current evoked by 1 μM muscarine was completely suppressed by 100 μM quinine, whereas 30 μM muscarine-induced inward currents were comprised of quinine-sensitive and -insensitive components. The electrophysiological and pharmacological properties of the muscarine-induced currents indicated that the quinine-sensitive and insensitive components are due to nonselective cation channel activation and TASK1 channel inhibition, respectively. Muscarine at 30 μM failed to induce any current in AM cells treated with muscarinic toxin 7 or genetically deleted of the M1 receptor. The KD value of VU0255035 against the muscarinic receptor mediating nonselective cation channel activation was 17.5 nM. These results indicate that the M1 receptor mediates nonselective cation channel activation as well as TASK1 channel inhibition.

Muscarinic receptor antagonists activate ERK-CREB signaling to augment neurite outgrowth of adult sensory neurons

A major cellular effector activated by G protein coupled receptors is extracellular signal-regulated kinase (ERK). The ERK signaling cascade regulates a variety of cellular processes including growth and proliferation. Both G protein and β-arrestin-mediated signaling lead to ERK activation by phosphorylation through different kinases. Recently, we have shown muscarinic acetylcholine type 1 receptor (M1R) antagonists, muscarinic toxin 7 (MT7) and pirenzepine, elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models. Thus, we tested the novel hypothesis that muscarinic antagonists could drive neurite outgrowth through altered M1R-ERK signaling. We have used two dimensional isoelectric focusing/SDS-PAGE combined with analysis using multiple phospho-epitope specific antibodies to study ERK1/2 phosphorylation and activation of its downstream nuclear effector cyclic response element binding protein (CREB). Activated CREB is known to exhibit neuroprotective and growth promoting effects. One hour of treatment with MT7 and pirenzepine activated ERK through M1R and induced a significant increase in levels of pCREB(S133) in cultured sensory neurons. Further, pharmacological blockade or siRNA based knockdown of ERK abolished the MT7 and pirenzepine mediated neuritogenic effect. In addition, we have shown drug-induced alterations of charged protein fractions that may possess additional post-translationally modified forms of ERK and CREB. For the first time we show that long-term treatment, e.g. 1 h, with muscarinic antagonists selective or specific for M1R can activate a biased β-arrestin dependent ERK-CREB signal cascade. Our study gives novel insight into muscarinic antagonist-mediated modulation of M1R-ERK-CREB signaling which could be exploited for therapy in neuropathic diseases.

Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons.

The muscarinic acetylcholine type 1 receptor (M1R) is a metabotropic G protein-coupled receptor. Knockout of M1R or exposure to selective or specific receptor antagonists elevates neurite outgrowth in adult sensory neurons and is therapeutic in diverse models of peripheral neuropathy. We tested the hypothesis that endogenous M1R activation constrained neurite outgrowth via a negative impact on the cytoskeleton and subsequent mitochondrial trafficking. We overexpressed M1R in primary cultures of adult rat sensory neurons and cell lines and studied the physiological and molecular consequences related to regulation of cytoskeletal/mitochondrial dynamics and neurite outgrowth. In adult primary neurons, overexpression of M1R caused disruption of the tubulin, but not actin, cytoskeleton and significantly reduced neurite outgrowth. Over-expression of a M1R-DREADD mutant comparatively increased neurite outgrowth suggesting that acetylcholine released from cultured neurons interacts with M1R to suppress neurite outgrowth. M1R-dependent constraint on neurite outgrowth was removed by selective (pirenzepine) or specific (muscarinic toxin 7) M1R antagonists. M1R-dependent disruption of the cytoskeleton also diminished mitochondrial abundance and trafficking in distal neurites, a disorder that was also rescued by pirenzepine or muscarinic toxin 7. M1R activation modulated cytoskeletal dynamics through activation of the G protein (Gα13) that inhibited tubulin polymerization and thus reduced neurite outgrowth. Our study provides a novel mechanism of M1R control of Gα13 protein-dependent modulation of the tubulin cytoskeleton, mitochondrial trafficking and neurite outgrowth in axons of adult sensory neurons. This novel pathway could be harnessed to treat dying-back neuropathies since anti-muscarinic drugs are currently utilized for other clinical conditions.

Muscarinic toxin 9: A novel peptide from Dendroaspis polylepis venom that selectively targets muscarinic type 2 receptor

Muscarinic toxin 9: A novel peptide from Dendroaspis polylepis venom that selectively targets muscarinic type 2 receptor

Muscarinic Antagonist Mediated Activation of ERK Signaling Depends on β‐arrestin Recruitment to Augment Axonal Outgrowth in Neurons

ObjectiveOne of the major cellular effectors activated by G protein coupled receptors is extracellular signal‐regulated kinase (ERK). The ERK signaling cascade regulates a variety of cellular processes including growth and proliferation. Both G protein and β‐arrestin mediated signaling pathways lead to ERK activation by phosphorylation through different kinases. Recently, we have shown muscarinic acetylcholine type 1 receptor (M1R) antagonists, muscarinic toxin 7 (MT7) and pirenzepine, induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models. Thus, we hypothesized that the neuritogenic effect of muscarinic antagonists was mediated by M1R‐ERK signaling.MethodologyWe have used extensive two dimensional isoelectric focusing/SDS‐PAGE combined with analysis using multiple phospho‐epitope specific antibodies to study ERK1/2 phosphorylation and activation of its downstream nuclear effector ‐ cyclic response element binding protein (CREB) in cultured adult rat primary dorsal root ganglion (DRG) neurons. Activation of CREB is known for neuroprotective and growth promoting effects. In addition, we have used CRISPR/Cas9 based β‐arrestin1/2 and GαS/L/Q/11/12/13 null HEK293 cell lines to dissect the downstream signaling events following antagonist binding to M1R.ResultsMT7 and pirenzepine‐mediated M1R‐ERK signaling induced a significant increase in activation‐loop specific phosphorylation (T202/Y204) of ERK1/2 and significantly elevated pCREB(S133) levels in cultured sensory neurons. Further, using M1R expressing β‐arrestin1/2 and GαS/L/Q/11/12/13 null HEK293 cell lines, we have shown that MT7 or pirenzepine mediated ERK1/2 activation is positively dependent on β‐arrestin recruitment to M1R but negatively regulated by G proteins. We have identified multiple charged fractions of ERK1/2 modulated by muscarinic antagonist treatment that is indicative of complex dynamics related to allosteric drug binding.ConclusionsOur study provides evidence that the neuritogenic effect of MT7 and pirenzepine may be mediated by selective blockade of M1R and downstream activation of ERK‐CREB signaling. We also give insights into allosteric drug binding and complex dynamics of ERK activation. In addition, our study also indicates that removal of negative regulation by G proteins on antagonist‐M1R‐ERK signaling could be exploited for improved therapy in neuropathic diseases.Support or Funding InformationSupported by CIHR grant # MOP‐130282 (PF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Muscarinic Acetylcholine Type 1 Receptor Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons: A Phenotype Rescued by Antagonist Treatment

ObjectiveThe muscarinic acetylcholine type 1 receptor (M1R) is a metabotropic G protein coupled receptor. Knockout of M1R or exposure to selective or specific receptor antagonists elevates neurite outgrowth in adult sensory neurons and blockade of associated cholinergic pathways is therapeutic in diverse models of peripheral neuropathy. We tested the hypothesis that endogenous M1R activation constrained neurite outgrowth via a negative impact on the cytoskeleton and subsequent mitochondrial trafficking and that this could be rescued by antagonist treatment.MethodologyWe overexpressed M1R and M1R DREADD (designer receptors exclusively activated by designer drugs) mutant in primary cultures of adult rat dorsal root ganglion (DRG) neurons and in tumor cell lines and studied the physiological and molecular consequences related to regulation of cytoskeletal/mitochondrial dynamics and neurite outgrowth. The M1R‐DREADD mutant contained two mutations of conserved orthosteric site residues that cause minimal responsiveness to ACh. In addition, we used CRISPR/Cas9 based Gα12/13 knockout HEK293 cells and siRNA mediated knockdown of Gα13 in cultured DRG neurons to study M1R Gα13 protein mediated tubulin cytoskeletal dynamics. No M1R ligand was added to the culture media.ResultsOverexpression of M1R by adult sensory neurons caused dissolution of the polymerized tubulin cytoskeleton, but not the actin network and significantly reduced neurite outgrowth. Conversely, over‐expression of a M1R‐DREADD mutant increased neurite outgrowth. These data suggest that endogenously released M1R ligand interacts with the M1R to suppress neurite outgrowth. M1R‐dependent disruption of the cytoskeleton significantly diminished mitochondrial abundance and trafficking in the distal neurites. Treatment with the specific M1R antagonist – muscarinic toxin 7 (MT7, 100 nM) or the selective M1R antagonist pirenzepine (1 μM) rescued cytoskeletal abnormalities caused by M1R overexpression by sequestering G‐proteins (Gα12/13) to the receptor. In addition, Gα12/13 null HEK293 cells exhibited increased polymerized tubulin and knockdown of Gα13 protected from tubulin cytoskeletal destabilization and restored growth in neurons overexpressing the M1R.ConclusionsOur study indicates that constitutively high expression of M1R destabilizes cytoskeletal dynamics through activation of Gα13 protein that increased tubulin de‐polymerization, impeded mitochondrial transport and reduced neurite outgrowth. This suggests a novel mechanism of M1R control of G protein‐dependent (Gα13) modulation of the tubulin cytoskeleton, mitochondrial trafficking and neurite outgrowth in axons of adult sensory neurons. Manipulation of this novel pathway could be used to treat dying‐back neuropathies since anti‐muscarinic drugs are currently utilized for other clinical conditions.Support or Funding InformationSupported by CIHR grant # MOP‐130282 (PF) and NIH grant NS081082 (NAC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Muscarinic type-1 receptors contribute to IK,ACh in human atrial cardiomyocytes and are upregulated in patients with chronic atrial fibrillation

BackgroundBasal and acetylcholine-gated inward-rectifier K+-currents (IK1 and IK,ACh, respectively) are altered in atrial fibrillation (AF). Gi-protein-coupled muscarinic (M) receptors type-2 are considered the predominant receptors activating IK,ACh. Although a role for Gq-coupled non-M2-receptor subtypes has been suggested, the precise regulation of IK,ACh by multiple M-receptor subtypes in the human atrium is unknown. Here, we investigated M1-receptor-mediated IK,ACh regulation and its remodeling in chronic AF (cAF). Methods and resultsM1-receptor mRNA and protein abundance were increased in atrial cardiomyocyte fractions and atrial homogenates from cAF patients, whereas M2-receptor levels were unchanged. The regulation of IK,ACh by M1-receptors was investigated in right-atrial cardiomyocytes using two applications of the M-receptor agonist carbachol (CCh, 2μM), with pharmacological interventions during the second application. CCh application produced a rapid current increase (Peak-IK,ACh), which declined to a quasi-steady-state level (Qss-IK,ACh). In sinus rhythm (Ctl) the selective M1-receptor antagonists pirenzepine (10nM) and muscarinic toxin-7 (MT-7, 10nM) significantly inhibited CCh-activated Peak-IK,ACh, whereas in cAF they significantly reduced both Peak- and Qss-IK,ACh, with no effects on basal inward-rectifier currents in either group. Conversely, the selective M1-receptor agonist McN-A-343 (100μM) induced a current similar to the CCh-activated current in Ctl atrial cardiomyocytes pretreated with pertussis toxin to inhibit M2-receptor-mediated Gi-protein signaling, which was abolished by MT-7. Computational modeling indicated that M1- and M2-receptors redundantly activate IK,ACh to abbreviate APD, albeit with predominant effects of M2-receptors. ConclusionOur data suggest that Gq-coupled M1-receptors also regulate human atrial IK,ACh and that their relative contribution to IK,ACh activation is increased in cAF patients. We provide novel insights about the role of non-M2-receptors in human atrial cardiomyocytes, which may have important implications for understanding AF pathophysiology.

Selective antagonism of muscarinic receptors is neuroprotective in peripheral neuropathy.

Sensory neurons have the capacity to produce, release, and respond to acetylcholine (ACh), but the functional role of cholinergic systems in adult mammalian peripheral sensory nerves has not been established. Here, we have reported that neurite outgrowth from adult sensory neurons that were maintained under subsaturating neurotrophic factor conditions operates under cholinergic constraint that is mediated by muscarinic receptor-dependent regulation of mitochondrial function via AMPK. Sensory neurons from mice lacking the muscarinic ACh type 1 receptor (M1R) exhibited enhanced neurite outgrowth, confirming the role of M1R in tonic suppression of axonal plasticity. M1R-deficient mice made diabetic with streptozotocin were protected from physiological and structural indices of sensory neuropathy. Pharmacological blockade of M1R using specific or selective antagonists, pirenzepine, VU0255035, or muscarinic toxin 7 (MT7) activated AMPK and overcame diabetes-induced mitochondrial dysfunction in vitro and in vivo. These antimuscarinic drugs prevented or reversed indices of peripheral neuropathy, such as depletion of sensory nerve terminals, thermal hypoalgesia, and nerve conduction slowing in diverse rodent models of diabetes. Pirenzepine and MT7 also prevented peripheral neuropathy induced by the chemotherapeutic agents dichloroacetate and paclitaxel or HIV envelope protein gp120. As a variety of antimuscarinic drugs are approved for clinical use against other conditions, prompt translation of this therapeutic approach to clinical trials is feasible.

Toxicovenomics and antivenom profiling of the Eastern green mamba snake (Dendroaspis angusticeps)

A toxicovenomic study was performed on the venom of the green mamba, Dendroaspis angusticeps. Forty-two different proteins were identified in the venom of D. angusticeps, in addition to the nucleoside adenosine. The most abundant proteins belong to the three-finger toxin (3FTx) (69.2%) and the Kunitz-type proteinase inhibitor (16.3%) families. Several sub-subfamilies of the 3FTxs were identified, such as Orphan Group XI (Toxin F-VIII), acetylcholinesterase inhibitors (fasciculins), and aminergic toxins (muscarinic toxins, synergistic-like toxins, and adrenergic toxins). Remarkably, no α-neurotoxins were identified. Proteins of the Kunitz-type proteinase inhibitor family include dendrotoxins. Toxicological screening revealed a lack of lethal activity in all RP-HPLC fractions, except one, at the doses tested. Thus, the overall toxicity depends on the synergistic action of various types of proteins, such as dendrotoxins, fasciculins, and probably other synergistically-acting toxins. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality. These antivenoms also showed a pattern of broad immunorecognition of the different HPLC fractions by ELISA and immunoprecipitated the crude venom by gel immunodiffusion. The synergistic mechanism of toxicity constitutes a challenge for the development of effective recombinant antibodies, as it requires the identification of the most relevant synergistic toxins. Biological significanceEnvenomings by elapid snakes of the genus Dendroaspis, collectively known as mambas, represent a serious medical problem in sub-Saharan Africa. The development of novel antivenoms and of recombinant neutralizing antibodies demands the identification of the most relevant toxins in these venoms. In this study, a bottom-up approach was followed for the study of the proteome of the venom of the Eastern green mamba, D. angusticeps. Forty-two different proteins were identified, among which the three-finger toxin (3FTx) family, characteristic of elapid venoms, was the most abundant, followed by the Kunitz-type proteinase inhibitor family. In addition, several other protein families were present in the venom, together with the nucleoside adenosine. No α-neurotoxins were identified within the family of 3FTxs in the venom of D. angusticeps, in contrast to the venom of Dendroaspis polylepis, in which α-neurotoxins are largely responsible for the toxicity. With one exception, HPLC fractions from D. angusticeps venom did not kill mice at the doses tested. This underscores that the toxicity of the whole venom is due to the synergistic action of various components, such as fasciculins and dendrotoxins, and probably other synergistically-acting toxins. Thus, the venoms of these closely related species (D. angusticeps and D. polylepis) seem to have different mechanisms to subdue their prey, which may be related to different prey preferences, as D. angusticeps is predominantly arboreal, whereas D. polylepis lives mostly in open bush country and feeds mainly on mammals. It is therefore likely that the predominant clinical manifestations of human envenomings by these species also differ, although in both cases neurotoxic manifestations predominate. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality in mice and showed a pattern of broad immunorecognition of the various venom fractions. It is necessary to identify the toxins responsible for the synergistic mode of toxicity in this venom, since they are the targets for the development of recombinant antibodies for the treatment of envenomings.

In Silico Analysis of Binding Interaction of Mamba Toxins with M4 and M2 Muscarinic Acetylcholine Receptors for Therapeutic Use in Alzheimer's Disease.

Muscarinic acetylcholine receptors are stimulated by the neurotransmitter acetylcholine and are involved in various functions across the human body. These receptors have surfaced for their potential use as targets in treatment of Alzheimer's disease. Muscarinic receptors have been reported to show binding interaction with various mamba toxins, such as dendrotoxins and muscarinic toxins that act as antagonists of these receptors. Therefore, in our study we have focused on the binding analysis of these mamba toxins with the M4 and M2 muscarinic acetylcholine autoreceptors for their potential use as targets in treating cognitive symptoms associated with Alzheimer's disease. A ligand dataset was developed that consisted of dendrotoxins and muscarinic toxins originating from various mamba species. Receptor dataset consisted of M4 and M2 muscarinic acetylcholine autoreceptors. Docking studies were performed using AutoDock 4.2 between these ligands with each receptor and further analysis was done using various computational tools. Docking experiments were performed and analyzed to check the binding compatibilities between mamba toxins and muscarinic acetylcholine autoreceptors. Detail analysis revealed that these ligands bind to active site residues of both receptors. Therefore by these in silico results, we suggest that the mamba toxins can be potential antagonists of the M4 and M2 muscarinic acetylcholine autoreceptors.

Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons

Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of −60 mV. Removal of extracellular Ca2+ markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca2+ was fully inhibited by removal of extracellular Na+, indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration–inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca2+/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca2+ from intracellular Ca2+ stores and calmodulin.The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

Structural determinants for the interactions between muscarinic toxin 7 and muscarinic acetylcholine receptors.

Muscarinic acetylcholine receptors (mAChRs) have five subtypes and play crucial roles in various physiological functions and pathophysiological processes. Poor subtype specificity of mAChR modulators has been an obstacle to discover new therapeutic agents. Muscarinic toxin 7 (MT7) is a natural peptide toxin with high selectivity for the M1 receptor. With three to five residues substituted, M3, M4, and M5 receptor mutants could bind to MT7 at nanomolar concentration as the M1 receptor. However, the structural mechanisms explaining MT7-mAChRs binding are still largely unknown. In this study, we constructed 10 complex models of MT7 and each mAChR subtype or its mutant, performed molecular dynamics simulations, and calculated the binding energies to investigate the mechanisms. Our results suggested that the structural determinants for the interactions on mAChRs were composed of some critical residues located separately in the extracellular loops of mAChRs, such as Glu4.56, Leu4.60, Glu/Gln4.63, Tyr4.65, Glu/Asp6.67, and Trp7.35. The subtype specificity of MT7 was attributed to the non-conserved residues at positions 4.56 and 6.67. These structural mechanisms could facilitate the discovery of novel mAChR modulators with high subtype specificity and enhance the understanding of the interactions between ligands and G-protein-coupled receptors.

Muscarinic Toxin 7, a Specific Acetylcholine Muscarinic Type 1 Receptor Antagonist, Corrects Deficits in Mitochondrial Bioenergetics in Cultured Sensory Neurons Derived from Diabetic Rodents

Muscarinic Toxin 7, a Specific Acetylcholine Muscarinic Type 1 Receptor Antagonist, Corrects Deficits in Mitochondrial Bioenergetics in Cultured Sensory Neurons Derived from Diabetic Rodents