Recombinant Expression of a Selective Blocker of M1 Muscarinic Receptors (original) (raw)
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The snake venom peptide Bj-PRO-7a is a M1 muscarinic acetylcholine receptor agonist
Cytometry Part A, 2011
Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensinconverting enzyme-independent mechanisms. Here, we show that Bj-PRO-7a (<EDGPIPP) specifically activates [Ca 21 ] i transients in CHO-M1 cells expressing heterologous rat M1 muscarinic subtype. The activation curve established by microfluorimetry in CHO-M1 cells using increasing concentrations of Bj-PRO-7a reached the maximum response in the presence of 3 lM Bj-PRO-7a (EC 50 5 0.25 6 0.07 lM). The variation observed by calcium imaging in these cells ranged from 52 to 1218 nM (EC 50 5 0.31 6 0.12 lM). [Ca 21 ] i responses in CHO-M1 cells were largely inhibited by pirenzepine, a specific M1 antagonist. Neural-differentiated P19 cells expressing endogenous M1 receptors were also responsive to Bj-PRO-7a application, whereas no such response was observed in undifferentiated P19 cells not expressing muscarinic receptors. As further support for its specific action on M1 receptors, the peptide did not activate M3 subtypes in transfected CHO cells. Our findings provide a novel M1 muscarinic receptor agonist that could be used for basic research and even for pharmacological applications. '
Muscarinic receptor subtype selective toxins
Life Sciences, 1997
The muscarinic acetylcholine receptors are monomeric proteins with seven hydrophobic, membrane spanning helices, and share a common evolutionary origin with the other members of the superfamily of membrane proteins known as sevenhelix receptors. The amino acid sequences of five different muscarinic acetylcholine receptors, called ml, m2, m3, m4 and m5 have been determined. The five subtypes are expressed to different extent in different tissues. A large number of low molecular ligands for muscarinic receptors are known, but they bind to all five subtypes of receptors and only a few of them have a slightly higher (five-six fold) affinity for one of the subtypes, e.g. pirenzepine for Ml (1) and tripitramine for M2 receptors (2). Several neurotoxins have been isolated from snake venoms and used as pharmacological tools. Mambas, African snakes of genus Dendroaspis, have toxins that recognize muscrinic receptors and some of these muscarinic toxins ate the most selective ligands for Ml and M4 receptors known to date.
Muscarinic Toxin 7 Selectivity Is Dictated by Extracellular Receptor Loops
Journal of Biological Chemistry, 2004
Muscarinic toxin 7 (MT7) is a mamba venom protein antagonist with extremely high selectivity for the M1 muscarinic acetylcholine receptor. To map the sites for the interaction of MT7 with muscarinic receptors we have used chimeric M1:M3 receptors and site-directed mutagenesis of the M3 and M4 receptor subtypes. Two Glu residues in M1, one in extracellular loop 2 and one in extracellular loop 3, were found to be important for the high affinity binding of MT7. Substitution of the corresponding Lys residues in the M3 receptor with Glu converted the M3 mutant to an MT7 binding receptor, albeit with lower affinity compared with M1. A Phe 3 Tyr substitution in extracellular loop 2 of M3 together with the 2 Glu mutations generated a receptor with an increased MT7 affinity (apparent K i ؍ 0.26 nM in a functional assay) compared with the M1 receptor (apparent K i ؍ 1.31 nM). The importance of the identified amino acid residues was confirmed with a mutated M4 receptor constructs. The results indicate that the high selectivity of MT7 for the M1 receptor depends on very few residues, thus providing good prospects for future design and synthesis of muscarinic receptor-selective ligands.
British Journal of Pharmacology, 2000
MT-7 (1 ± 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M 1 receptor, inhibited the acetylcholine (ACh)-stimulated [ 35 S]-guanosine-5'-O-(3-thio)triphosphate ([ 35 S]-GTPgS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M 1 receptor subtype. 2 MT-7 failed to aect the ACh-stimulated [ 35 S]-GTPgS binding in membranes of CHO cells expressing either the M 2 , M 3 or M 4 receptor subtype. 3 In N1E-115 neuroblastoma cells endogenously expressing the M 1 and M 4 receptor subtypes, MT-7 (0.3 ± 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to aect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. 4 In both CHO/M 1 and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist eect with minimal changes in the agonist EC 50 value. 5 In CHO/M 1 cell membranes, MT-7 (0.05 ± 25 nM) reduced the speci®c binding of 0.05, 1.0 and 15 nM [ 3 H]-N-methylscopolamine ([ 3 H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [ 3 H]-NMS by about 5 fold. 6 CHO/M 1 cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [ 3 H]-NMS binding for at least 8 h at 308C. 7 It is concluded that MT-7 acts as a selective noncompetitive antagonist of the muscarinic M 1 receptors by binding stably to an allosteric site.
Two-step binding of green mamba toxin to muscarinic acetylcholine receptor
FEBS Letters, 1994
The mechanism of binding of toxin MT2 from venom of green mamba Dendroaspis angusticeps to muscarinic acetylcholine receptors from rat cerebral cortex was investigated by studying the kinetics of the toxin-receptor interaction. The muscarinic antagonist N-methyl-['Hjscopolamine was used as a 'reporter' ligand. Evidence for a mechanism of toxin-receptor interaction comprising at least two steps was obtained. Such a mechanism increases the potency of the toxin. The first step was fast with no competition between the toxin and the antagonist. The second step was slow with formation of a more stable toxin-receptor complex and inhibition of the antagonist binding. It is proposed that the snake toxin is a muscarinic agonist of slow action.
Muscarinic toxin-like proteins from cobra venom
European Journal of Biochemistry, 2000
Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55±74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30±34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only.
Neurochemistry International, 1992
Two new polypeptides were isolated and purified from the venom of the snake Dendroaspis angusticeps, which also contains other neuroactive peptides such as Dendrotoxins and Fasciculins. The amino acid composition of the peptides was determined and the first 10 amino acids from the MTX2 Nterminal fragment were sequenced. The so-called muscarinic toxins (MTX1 and MTX2) have been shown to inhibit the specific binding of 3H]QNB (0.15 nM), [3H]pZ (2.5 nM) and [3H]oxoM (2 nM) to bovine cerebral cortex membranes by 60, 88 and 82% respectively. In contrast, they caused only a 30% blockade of the [3H]QNB specific binding to similar membrane preparations from the brainstem. The Hill number for the [3H]pZ binding inhibition by the putative muscarinic toxin MTX2 was 0.95 suggesting homogeneity in the behaviour of the sites involved. The data from [3H]oxoM binding gave a Hill number of 0.83. The decreases in the specific binding involved increases in KD for the three different ligands (8-fold for [3H]QNB, 4-fold for [3H]pZ and 3.5-fold for [3H]oxoM) without significant changes in Bmax, except for a slight decrease in the [3H]oxoM binding sites (-19%); such results suggest that there may be a competitive inhibition between the MTXs and these ligands. The Ki for MTX2/[3H]pZ was 22.58+3.52 nM; for MTX2/[3H]oxoM, 144.9_+21.07 nM and for MTX2/[3H]QNB, 134.98_+ 18.35 nM.
Snake toxins with high selectivity for subtypes of muscarinic acetylcholine receptors 2000
There are five subtypes of muscarinic acetylcholine receptors (M 1 to M 5 ) which control a large number of physiological processes, such as the function of heart and smooth muscles, glandular secretion, release of neurotransmitters, gene expression and cognitive functions as learning and memory. A selective ligand is very useful for studying the function of a subtype in presence of other subtypes, which is the most common situation, since a cell or an organ usually has several subtypes. There are many non-selective muscarinic ligands, but only few selective ones. Mambas, African snakes of genus Dendroaspis have toxins, muscarinic toxins, that are selective for M 1 , M 2 and M 4 receptors. They consist of 63-66 amino acids and four disulfides which form four loops. They are members of a large group of snake toxins, three-finger toxins; three loops are extended like the middle fingers of a hand and the disulfides and the shortest loop are in the palm of the hand. Some of the toxins target the allosteric site which is located in a cleft of the receptor molecule close to its extracellular part. A possible explanation to the good selectivity is that the toxins bind to the allosteric site, but because of their size they probably also bind to extracellular parts of the receptors which are rather different in the various subtypes. Some other allosteric ligands also have good selectivity, the alkaloid brucine and derivatives are selective for M 1 , M 3 and M 4 receptors. Muscarinic toxins have been used in several types of experiments. For instance radioactively labeled M 1 and M 4 selective toxins were used in autoradiography of hippocampus from Alzheimer patients. One significant change in the receptor content was detected in one region of the hippocampus, dentate gyrus, where M 4 receptors were reduced by 50% in patients as compared to age-matched controls. Hippocampus is essential for memory consolidation. M 4 receptors in dentate gyrus may play a role, since they decreased in Alzheimers disease which destroys the memory. Another indication of the role of M 4 receptors for memory is that injection of the M 4 selective antagonist muscarinic toxin 3 (M 4 -toxin 1) into rat hippocampus produced amnesia. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS snake toxins / muscarinic receptor subtypes / selectivity * Correspondence and reprints to Lindsbergsgatan 11A,