Muscarinic Toxin Research Articles

Muscarynic metabotropic receptor M4 modulates the hippocampal CA1 LTP possibly through local GABAergic interneurons

Muscarinic Cholinergic receptors of the subtype M4 are G protein coupled receptors that activate Gi reducing AMPc concentration on the cell and closing K+ channels, causing hyperpolarization of the cell membrane and reduction of neurotransmitter release [1]. Previous works from our laboratory using the very selective Muscarinic Toxin 3 (MT3) to block M4 receptors showed that infusion of this substance into the dorsal hippocampus impairs memory consolidation [2] and facilitates memory retrieval [3], suggesting an important role for M4 modulation of CA1 circuitry in memory processes. Also, local application of this toxin in hippocampal slices was able to impair long-term potentiation (LTP) [4]. One hypothesis raised to explain these findings suggests that plastic changes taking place in the CA1 circuitry involves differential control of local excitatory cells and inhibitory interneurons. Since changes in LTP can be elicited by different mechanisms, the objective of this work was to verify, through an analog of the pharmacological infusion used in the behavioral studies, the effect of MT3 on LTP and the activity of inhibitory interneurons as possible targets of this cholinergic action. Thus, we recorded fEPSP from Schaffer collaterals at hippocampal CA1 area of rats anesthetized under urethane. After setting the position and the stimulation intensity to obtain 50% of maximum fEPSP, we recorded 10 min baseline followed by drug infusion (0.5μl, 20μl/h) through a delicate cannula glued to the recording electrode at the contralateral side of stimulae. We infused either MT3 (2.0μg), baclofen (0.1μg), MT3+baclofen (same doses), or their vehicles. After 15min, we applied a high frequency stimulation (HFS) protocol (10 trains at 0.5Hz of 20 pulses 100Hz) and recorded the fEPSP for at least 90min. None of the treatments affected the baseline excitability. Our results indicate that: MT3 infusion 15min before HFS was able to block LTP, consistent with our previous finding with MT3 applied 2min before HFS [4], and baclofen, a GABAB receptor agonist in a subeffective dose, was able to prevent this MT3 effect. Considering that hippocampal GABAB receptors are mainly presynaptic and mostly located in GABAergic interneurons, and that its activation leads to cell hyperpolarization / reduction of GABA release, our results suggest that the M4 receptors relevant to those behavioral responses may be colocalized with GABAB receptors in the CA1 interneurons. Further studies are necessary to verify this hypothesis.

Ophiophagus hannah Venom: Proteome, Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

Polypharmacology profiles and phylogenetic analysis of three-finger toxins from mamba venom: case of aminergic toxins.

Composition of mamba's venom is quite atypical and characterized by the presence of a large diversity of three-finger fold toxins (3FTx) interacting with various enzymes, receptors and ion channels. In particular, 3FTx from mambas display the unique property to interact with class A GPCRs, sometimes with a high affinity and selectivity. A screening of five of these toxins (MT1, MT3, MT7, ρ-Da1a and ρ-Da1b) on 29 different subtypes of bioaminergic receptors, using competition binding experiments, highlights the diversity of their pharmacological profiles. These toxins may display either absolute selectivity for one receptor subtype or a polypharmacological property for various bioaminergic receptors. Nevertheless, adrenoceptor is the main receptor family targeted by these toxins. Furthermore, a new receptor target was identified for 3FTx and toxins in general, the ρ-Da1b interacting competitively with the human dopamine D3 receptor in the micromolar range. This result expands the diversity of GPCRs targeted by toxins and more generally highlights the multipotent interacting property of 3FTx. Phylogenic analyzes of these toxins show that muscarinic, adrenergic and dopaminergic toxins may be pooled in one family called aminergic toxins, this family coming probably from a specific radiation of ligands present in mamba venoms.

Mass Spectrometric Characterization of Disulfide Bridges in Snake Venom Proteins

Venoms fromsnakes, spiders, scorpions etc.offer anAladdin’s cave of proteins and peptides for drug discovery. Within the venom of these species are numerous proteins and enzymes designed to kill or immobilise prey and aid digestion, including neurotoxins, cardiotoxins and muscarinic toxins. The individual proteins can be isolated (or produced recombinantly) and used for therapeutic effect. Disulfide bridges are covalent bonds formed between the thiol groups of two cysteine residues. Many venom proteins contain a proportionally large number of such bridges, which play a fundamental role in defining their structure and specialized functionality. For many proteins, disulfide assignment can be achieved fairly simply by mass spectrometry (MS) analysis of bridged fragments following proteolytic digestion under non-reducing conditions. In venom proteins, elucidation of disulfide connectivities is often much more challenging due to the interwoven arrangement of bridges, which means that a simple proteolytic digest will not cut the protein in such a way that the connectivity can be determined unambiguously. Thus a variety of techniques are required to yield fragments that will reveal the disulfide connectivity. At HES-SO Valais, recombinant snake venom proteins are being developed for use as new drugs. Part of the analytical strategy is to verify that disulfide bridges are correctly formed. To confirm their number, a high mass accuracy QTOF mass spectrometer is used to observe the mass difference of 1 Da between a disulfide bonded and free cysteine. The presence of disulfide isoforms (proteins with identical sequence but different disulfide connectivities) is determined via MS following their separation with RP-(U) HPLC. To assign bridges, proteins are digested using carefully selected combinations of proteolytic enzymes such as trypsin and pepsin to give proteolytic fragments which consist of several peptide chains linked by disulfide bridges. MS/ MS with collision-induced dissociation is then used to cleave along the polypeptide backbone to give peptide fragments joined together by single disulfides, from which the bonding pattern can be deduced. This enables us to study challenging proteins containing interwoven disulfides and vicinal cysteines as are often an occurrence in venom proteins. Verifying disulfide bonding in venom protein-based drugs is a key element in assuring Active Pharmaceutical Ingredient safety and efficacy.

Functional Activation of G-Proteins Coupled With Muscarinic Acetylcholine Receptors in Rat Brain Membranes

The functional activation of Gi/o proteins coupled to muscarinic acetylcholine receptors (mAChRs) was investigated with the conventional guanosine-5'-O-(3-[(35)S]thio) triphosphate ([(35)S]GTPγS) binding assay in rat brain membranes. The most efficacious stimulation elicited by acetylcholine or carbachol (CCh) was obtained in striatal membranes. The pharmacological properties of mAChR-mediated [(35)S]GTPγS binding determined with a series of muscarinic agonists and antagonists were almost identical among the three brain regions investigated, i.e., cerebral cortex, hippocampus, and striatum, except for the apparent partial agonist effects of (αR)-α-cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate (J 104129) observed only in the hippocampus, but not in the other two regions. Among the muscarinic toxins investigated, only MT3 attenuated CCh-stimulated [(35)S] GTPγS binding. The highly selective allosteric potentiator at the M4 mAChR subtype, 3-amino-N-[(4-chlorophenyl)methyl]-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (VU 10010), shifted the concentration-response curve for CCh leftwards as well as upwards. On the other hand, neither thiochrome nor brucine N-oxide was effective. The increases induced by CCh and 5-HT were essentially additive, though not completely, indicating that the mAChRs and 5-HT1A receptors were coupled independently to distinct pools of Gi/o proteins. Collectively, all of the data suggest that functional activation of Gi/o proteins coupled to mAChRs, especially the M4 subtype, is detectable by means of CCh-stimulated [(35)S]GTPγS binding assay in rat discrete brain regions.

Proteomic analysis of Moroccan cobra Naja haje legionis venom using tandem mass spectrometry

The proteome of the venom of Naja haje legionis, the only medically important elapid species in Morocco, has been elucidated by using a combination of proteomic techniques that includes size exclusion chromatography, reverse-phase HPLC, Tricine/SDS-Page, tryptic digestion, Q-TOF tandem mass spectrometry and database search. The sequence analysis of venom fractions revealed a highly complex venom proteome which counts a total of 76 proteins identified from database that can be assigned into 9 proteins families. We report the identification of: cobra venom factor (CVF), l-amino-acid oxidases (LAAO), acetylcholinesterase (AChE), snake venom metalloproteinases (SVMP), cysteine rich secretory proteins (CRISP), venom nerve growth factor (vNGF), phospholipases A2 (PLA2), vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cardiotoxins and cytotoxins. Comparison of these proteins showed high sequence homology with proteins from other African and Asian cobras. Further works are needed to assess the contribution of individual toxins in venom toxicity. Naja haje legionis is one of the medically important snakes implicated in the pathogenesis of snake bite in Morocco. The absence of information about venom composition and clinical manifestations of envenomation by this cobra represents an obstacle for the management of this environmental disease in the country. The elucidation of Moroccan cobra venom composition will provide a reasonable guidance for clinician to understand the pathophysiological conditions associated with cobra envenomation and the elaboration of better management strategies.

Aminergic toxins: What they are, what they do and what we can do with

Aminergic toxins: What they are, what they do and what we can do with

Orthosteric Binding of ρ-Da1a, a Natural Peptide of Snake Venom Interacting Selectively with the α1A-Adrenoceptor

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of 3H-prazosin or 125I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of 3H-prazosin or 125I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca2+ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D1063.32A and the S1885.42A/S1925.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F862.64A, F2886.51A and F3127.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F862.64 was identified as a key interaction point for 125I-HEAT, as the variant F862.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F862.64, F2886.51 and F3127.39) and agonist (F2886.51 and F3127.39) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F862.64, which appears to be important also for HEAT binding.

Human organic cation transporter 2 (hOCT2): Inhibitor studies using S2-hOCT2 cells

Highly expressed in kidney and located on the basolateral membrane, human organic cation transporter 2 (hOCT2) can transport various compounds (i.e. drugs and toxins) into the proximal tubular cell. Using cultured proximal tubule cells stably expressing hOCT2 (i.e. S2-hOCT2 cells), we sought to probe different compound classes (e.g. analgesics, anti-depressants, anti-psychotics, disinfectant, herbicides, insecticides, local anesthetic, muscarinic acetylcholine receptor antagonist, sedatives, steroid hormone, stimulants and toxins) for their ability to inhibit 14C-TEA uptake, a prototypical OCT2 substrate. Aconitine, amitriptyline, atropine, chlorpyrifos, diazepam, fenitrothion, haloperidol, lidocaine, malathion, mianserin, nicotine and triazolam significantly inhibited 14C-TEA uptake; IC50 values were 59.2, 2.4, 2.0, 20.7, 32.3, 13.2, 32.5, 104.6, 71.1, 17.7, 52.8 and 65.5μM, respectively. In addition, aconitine, amitriptyline, atropine, chlorpyrifos, fenitrothion, haloperidol, lidocaine, and nicotine displayed competitive inhibition with Ki values of 145.6, 2.5, 2.4, 24.8, 16.9, 51.6, 86.8 and 57.7μM, respectively. These in vitro data support the notion that compounds pertaining to a wide variety of different drug classes have the potential to decrease renal clearance of drugs transported via hOCT2. Consequently, these data warrant additional studies to probe hOCT2 and its role to influence drug pharmacokinetics.

Pharmacological characterization of M1 muscarinic acetylcholine receptor-mediated Gq activation in rat cerebral cortical and hippocampal membranes

This study aimed to pharmacologically characterize the response derived from functional activation of Gq proteins coupled with native muscarinic acetylcholine receptors in rat cerebral cortex and hippocampus. Rat cerebral cortical and hippocampal membranes were prepared, and the effects of a range of mAChR agonists and antagonists, allosteric modulators, and muscarinic toxins were determined by an antibody-capture scintillation proximity assay combined with [(35)S]GTPγS binding, using the anti-Gαq antibody sc-393. Increased specific [(35)S]GTPγS binding, elicited by carbachol (CCh), was selectively inhibited by the muscarinic toxin MT7, and was resistant to membrane pretreatment with N-ethylmaleimide, indicating that the response derived exclusively from Gαq, selectively coupled with the M1 mAChR. In addition to CCh, many mAChR agonists, including oxotremorine, arecholine, and methacholine, stimulated binding in a concentration-dependent manner with varied potencies and efficacies. The intrinsic activities of partial M1 mAChR agonists in the present study were generally lower than previously reported in M1-expressing cells. Xanomeline and N-desmethylclozapine had negligible or minimal agonist properties. CCh-stimulated [(35)S]GTPγS binding to Gαq was inhibited by mAChR antagonists, including scopolamine, ipratropium, atropine, 4-DAMP, pirenzepine, and AF-DX 116, with a rank order of potency consistent with previous studies of M1-expressing cells. There was a highly significant correlation between the potencies of 13 agonists and 19 antagonists in the cerebral cortex and hippocampus. The effects of several allosteric mAChR modulators were also investigated. These data provide a comprehensive pharmacological profile of the Gq-coupled M1 mAChR subtype natively expressed at physiological levels in rat cerebral cortex and hippocampus.

Crystallization of recombinant green mamba ρ-Da1a toxin during a lyophilization procedure and its structure determination.

ρ-Da1a toxin from eastern green mamba (Dendroaspis angusticeps) venom is a polypeptide of 65 amino acids with a strong affinity for the G-protein-coupled α(1A)-adrenoceptor. This neurotoxin has been crystallized from resolubilized lyophilized powder, but the best crystals grew spontaneously during lyophilization. The crystals belonged to the trigonal space group P3(1)21, with unit-cell parameters a = b = 37.37, c = 66.05 Å, and diffracted to 1.95 Å resolution. The structure solved by molecular replacement showed strong similarities to green mamba muscarinic toxins.

New α-adrenergic property for synthetic MTβ and CM-3 three-finger fold toxins from black mamba

Despite their isolation more than fifteen years ago from the venom of the African mamba Dendroaspis polylepis, very few data are known on the functional activity of MTβ and CM-3 toxins. MTβ was initially classified as a muscarinic toxin interacting non-selectively and with low affinity with the five muscarinic receptor subtypes while no biological function was determined for CM-3. Recent results highlight the multifunctional activity of three-finger fold toxins for muscarinic and adrenergic receptors and reveal some discrepancies in the pharmacological profiles of their venom-purified and synthetic forms. Here, we report the pharmacological characterization of chemically-synthesized MTβ and CM-3 toxins on nine subtypes of muscarinic and adrenergic receptors and demonstrate their high potency for α-adrenoceptors and in particular a sub-nanomolar affinity for the α1A-subtype. Strikingly, no or very weak affinity were found for muscarinic receptors, highlighting that pharmacological characterizations of venom-purified peptides may be risky due to possible contaminations. The biological profile of these two homologous toxins looks like that one previously reported for the Dendroaspis angusticeps ρ-Da1a toxin. Nevertheless, MTβ and CM-3 interact more potently than ρ-Da1a with α1B- and α1D-AR subtypes. A computational analysis of the stability of the MTβ structure suggests that mutation S38I, could be involved in this gain in function.

M1 is a major subtype of muscarinic acetylcholine receptors on mouse colonic epithelial cells

Muscarinic acetylcholine receptors (mAChRs) are major regulators of gut epithelial functions. However, the precise subtype composition has not been clarified. We characterized the pharmacological profile of mAChRs on mouse colonic crypts, employing [(3)H]-N-methyl scopolamine chloride as a radioligand and several subtype-selective chemicals, and the functional aspect by measuring short-circuit current (I sc) in Ussing chambers and by evaluating MAP kinase phosphorylation in mouse colonic mucosal sheets. The mAChRs were detected on the crypts (K d=163.2±32.3pM, B max=47.3±2.6fmol/mg of total cell protein). Muscarinic toxin 7 (MT-7, M1 subtype selective) gave a displacement curve with high affinity, but there was a part insensitive to MT-7 (18.8±0.4% of the total specific binding). The MT-7-insensitive component was displaced completely by darifenacin (M3 selective) with high affinity. ACh induced an increase in I sc, which was significantly enhanced by MT-7 but was completely inhibited by darifenacin or atropine. Colitis induction resulted in a significant decrease in the density of mAChRs, which occurred mainly in the MT-7-sensitive component (M1 subtype). Immunological experiments exhibited a reduction of M1 but not of M3 signal after colitis induction. Muscarinic stimulation induced an increase in MAP kinase phosphorylation, which was completely suppressed by MT-7 and was attenuated by inflammation, in mouse colonic epithelium. These results suggest that mAChRs in mouse colonic epithelial cells consist of two subtypes, M1 (80%) and M3 (20%). The major M1 subtype was likely to regulate epithelial chloride secretion negatively and was susceptible to inflammation and may be relevant to inflammatory gut dysfunction.

Muscarinic receptor M3 mediates human gallbladder contraction through voltage-gated Ca2+ channels and Rho kinase

Objective. Muscarinic receptors mediate contraction of the human gallbladder through unclear receptor subtypes. The aim of the present study was to characterize muscarinic acetylcholine receptors mediating contraction of the human gallbladder. Materials and methods. Contraction of human gallbladder muscle strips caused by agonists carbachol and muscarine was measured and the inhibition of carbachol-induced contraction by muscarinic receptor antagonists was evaluated. Reverse transcription polymerase chain reaction was performed to determine the existence of muscarinic receptor subtypes. Results. Carbachol and muscarine caused concentration-dependent contraction of gallbladder strips. Four receptor antagonists, including atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), methoctramine, and pirenzepine, inhibited the carbachol-induced contraction. The relative inhibitory potency of these receptor antagonists was atropine > 4-DAMP > methoctramine > pirenzepine. The antagonist affinity estimates (pA2 values) correlated with the known affinities at M3, M4, and M5 muscarinic receptors. In addition, the M4-selective antagonist muscarinic toxin 3 did not inhibit and the M5-selective positive allosteric modulator VU0238429 did not potentiate carbachol-induced gallbladder contraction. This suggests that M3 muscarinic receptors mediate the muscarinic response predominantly. The contractile response of carbachol was attenuated by the voltage-gated Ca2+ channel inhibitor nifedipine and Rho-kinase inhibitor H-1152, but not affected by protein kinase C inhibitor chelerythrine. This implies the involvement of voltage-gated Ca2+ channel and Rho kinase but not protein kinase C. Conclusions. These results suggest a major role of M3 muscarinic receptors mediating the human gallbladder contraction through voltage-gated Ca2+ channels and Rho kinase. M3-selective muscarinic receptor antagonists could be of therapeutic importance in the treatment of biliary motility disorders.

Muscarinic Antagonist Control of Myopia: Evidence for M4and M1Receptor-Based Pathways in the Inhibition of Experimentally-Induced Axial Myopia in the Tree Shrew

The broadband muscarinic antagonist atropine is effective in stopping the progression of myopia in animals and humans. The partially selective M(1)/M(4) antagonist pirenzepine also slows progression of myopia, although not as effectively as atropine. Due to the supra maximal doses utilized in these studies, it is unclear if this antimyopia effect occurs through a receptoral-based mechanism, and if so, which receptors are involved. Studies in chicks indicate the involvement of the M(4) muscarinic receptor. The current study investigated the effect of the highly selective muscarinic antagonists Muscarinic Toxin 3 (MT3) (M(4) selective) and Muscarinic Toxin 7 (MT7) (M(1) selective) on experimental myopia in a mammalian model. Tree shrews (n = 23) underwent daily intravitreal injections of MT3, MT7, or vehicle (phosphate buffered saline) for five days in the treated eye, combined with deprivation of vision with a translucent occluder (MD). The contralateral eye was unocccluded and underwent intravitreal injections of vehicle for the same period. Two additional groups (n = 10) underwent daily intravitreal injections of MT7 or vehicle for 10 days in the treated eye combined with negative lens (-9.5 diopter [D]) defocus (LIM). The control eye was injected with saline and wore a plano lens. Both MT3 and MT7 treatment reduced the development of deprivation-induced myopia (treated-control eye [T-C]; vehicle-MD; -4.3 ± 0.6 D versus MT3-MD; -0.7 ± 0.2 D and MT7-MD; -0.7 ± 0.4 D; P < 0.001). MT7 treatment was effective at inhibiting lens-induced myopia (T-C; vehicle-LIM; -4.6 ± 0.5 D versus MT7-LIM; 0.2 ± 0.2 D; P < 0.05). The findings demonstrate that inhibition of form-deprivation myopia by muscarinic antagonists involves both M(4) and M(1) muscarinic receptor signaling pathways in mammals.

Muscarinic receptors acting at pre- and post-synaptic sites differentially regulate dopamine/DARPP-32 signaling in striatonigral and striatopallidal neurons

Muscarinic receptors, activated by acetylcholine, play critical roles in the functional regulation of medium spiny neurons in the striatum. However, the muscarinic receptor signaling pathways are not fully elucidated due to their complexity. In this study, we investigated the function of muscarinic receptors in the striatum by monitoring DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) phosphorylation at Thr34 (the PKA-site) using mouse striatal slices. Treatment of slices with a non-selective muscarinic receptor agonist, oxotremorine (10 μM), rapidly and transiently increased DARPP-32 phosphorylation. The increase in DARPP-32 phosphorylation was completely abolished either by a dopamine D1 receptor antagonist (SCH23390), tetrodotoxin, genetic deletion of M5 receptors, muscarinic toxins for M1 and M4 receptors, or 6-hydroxydopamine lesioning of dopaminergic neurons, whereas it was enhanced by nicotine. Analysis in D1-DARPP-32-Flag/D2-DARPP-32-Myc transgenic mice revealed that oxotremorine increases DARPP-32 phosphorylation selectively in D1-type/striatonigral, but not in D2-type/striatopallidal, neurons. When D1 and D2 receptors were blocked by selective antagonists to exclude the effects of released dopamine, oxotremorine increased DARPP-32 Thr34 phosphorylation only in D2-type/striatopallidal neurons. This increase required activation of M1 receptors and was dependent upon adenosine A2A receptor activity. The results demonstrate that muscarinic receptors, especially M5 receptors, act at presynaptic dopaminergic terminals, regulate the release of dopamine in cooperation with nicotinic receptors, and activate D1 receptor/DARPP-32 signaling in the striatonigral neurons. Muscarinic M1 receptors expressed in striatopallidal neurons interact with adenosine A2A receptors and activate DARPP-32 signaling.

225. Proteomic Analysis and Pharmacological Activities of the Venom of the Moroccan Cobra Naja haje legionis.

Background: In Morocco, envenomation by snake bites poses a serious problem to public health. The cobra Naja haje legionis is endemic of the country and one of the most dangerous species known. In this work, we report the proteomic and pharmacological characterizations of biologically active proteins from the venom of this species. Methods: The various proteins in the crude venom were fractionated and analyzed by a combination of chromatographic separations and proteomic analysis techniques such as gel filtration, RP-HPLC, 1D electrophoresis, in-gel digestion, tandem mass spectrometry and protein database search. The pharmacological properties of Naja haje legionis venom were assessed using in vitro preparations using rodents and chicks. Results: Our venomic strategy allowed the identification of 64 proteins and peptides from known database which can classified into 17 families according to their biological activities. We were able to identify cobra venom factor, L-aminoacid oxidases, acetylcholinesterase, metalloproteinase, disintegrin, cysteine-rich secretory proteins, nerve growth factor, phospholipases A2, vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cytotoxins and cardiotoxins. Pharmacological tests showed that Naja haje legionis venom contained neurotoxic activities inducing irreversible blockage of neuromuscular transmission in both rodent and chick nerve-muscle preparations. This venom also exhibit myotoxic and cardiotoxic activities. Conclusions: These results showed the potency and the complexity of the various proteins present in the venom of Naja haje legionis,which present a very similar pattern to other cobra venoms. The contribution of the different components in venom toxicity deserves further investigations.

Characterization of α2B-adrenoceptor ligand binding in the presence of Muscarinic toxin α and delineation of structural features of receptor binding selectivity

Muscarinic toxin α (MTα), a peptide isolated from the venom of the African black mamba, was recently found to selectively antagonize the human α2B-adrenoceptor. To gain more information about the binding of this peptide toxin, we studied the properties of the [3H]UK14,304 agonist and the [3H]MK-912 antagonist binding to the α2B-adrenoceptor in the presence of MTα. In equilibrium binding experiments, MTα decreased the binding of the orthosteric ligands, but failed to completely displace these. This effect of MTα was due to noncompetitive inhibition of Bmax without change in radioligand affinity. On the contrary, cellular signaling via the α2B-adrenoceptor could be titrated to zero despite the incomplete receptor blockade. To locate binding sites for MTα on the receptor protein, we generated chimeric receptors of α2B- and α2A- or α2C-adrenoceptors. Data based on these constructs revealed the extracellular loop two (ECL2) as the structural entity that enables MTα binding. Cumulative exchange of parts of ECL2 of α2B for α2A-adrenoceptor sequence resulted in a gradual decrease in the affinity for MTα, indicating that MTα binds to the α2B-adrenoceptor through multiple sites dispersed over the whole ECL2. Together the results suggest that binding of MTα to the α2B-adrenoceptor occludes orthosteric ligand access to the binding pocket. Putative homomeric receptor complexes as factors underlying the apparent noncompetitivity are also discussed.

Glycosylphosphatidylinositol (GPI)-anchoring of mamba toxins enables cell-restricted receptor silencing

Muscarinic toxins (MTs) are snake venom peptides found to selectively target specific subtypes of G-protein-coupled receptors. In here, we have attached a glycosylphosphatidylinositol (GPI) tail to three different toxin molecules and evaluated their receptor-blocking effects in a heterologous expression system. MT7–GPI remained anchored to the cell surface and selectively inhibited M1 muscarinic receptor signaling expressed in the same cell. To further demonstrate the utility of the GPI tail, we generated MT3- and MTα-like gene sequences and fused these to the signal sequence for GPI attachment. Functional assessment of these membrane-anchored toxins on coexpressed target receptors indicated a prominent antagonistic effect. In ligand binding experiments the GPI-anchored toxins were found to exhibit similar selection profiles among receptor subtypes as the soluble toxins. The results indicate that GPI attachment of MTs and related receptor toxins could be used to assess the role of receptor subtypes in specific organs or even cells in vivo by transgenic approaches.

Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein

Muscarinic toxin 7 (MT7) is a mamba venom peptide that binds selectively to the M1 muscarinic acetylcholine receptor. We have previously shown that the second (ECL2) and third (ECL3) extracellular loops of the M1 receptor are critically involved in binding the peptide. In this study we used a mutagenesis approach on the M5 subtype of the receptor family to find out if this possesses a similar structural architecture in terms of toxin binding as the M1 receptor. An M5 receptor construct (M5-E175Y184E474), mutated at the formerly deciphered critical residues on ECL2 and 3, gained the ability to bind MT7, but with rather low affinity as determined in a functional assay (apparent Ki = 24 nM; apparent Ki for M1 = 0.5 nM). After screening for different domains and residues, we found a specific residue (P179 to L in M5) in the middle portion of ECL2 that was necessary for high affinity binding of MT7 (M5-EL179YE, apparent Ki = 0.5 nM). Mutation of P179 to A confirmed a role for the leucine side chain in the binding of MT7. Together the results reveal new binding interactions between receptors and the MT7 peptide and strengthen the hypothesis that ECL2 sequence is of utmost importance for MT binding to muscarinic receptors.