Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

Abstract Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

摘要 背景：本文报道了从条纹珊瑚蛇（Micrurus lemniscatus）毒液中分离得到的一种具有磷脂酶A2活性的神经毒素Mlx-8，该毒素对毒蕈碱型乙酰胆碱受体（muscarinic acetylcholine receptors, mAChRs）具有亲和力。方法：本研究对Mlx-8进行了纯化、分子质量测定、部分氨基酸序列测序、磷脂酶A2活性检测，以及选择性毒蕈碱配体[3H]QNB结合抑制实验，同时检测了其对大鼠海马组织中总[3H]肌醇磷酸积累的抑制作用。结果：通过高效液相色谱（HPLC）共收集到31个组分，本研究使用的毒素为Mlx-8。Mlx-8的分子质量为13.628 Da。经埃德曼降解（Edman degradation）得到其部分氨基酸序列：NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT。Mlx-8具有磷脂酶A2酶活性。通过[3H]QNB竞争结合实验，测定了Mlx-8与M1型选择性毒蕈碱拮抗剂哌仑西平（pirenzepine）在海马膜中的pKi值。分析二者的置换曲线得到的pKi值分别为7.32±0.15（n=4）和5.84±0.18（n=4），上述结果表明Mlx-8对mAChRs具有亲和力。当1 μM Mlx-8与200 μM二乙基二硫代氨基甲酸酯（DEDA，一种磷脂酶A2抑制剂）共同孵育时，其对海马膜中[3H]QNB结合的抑制能力未受影响，这提示毒液的磷脂酶A2活性被抑制后，并未改变其置换[3H]QNB结合的能力。此外，当使用0.1 μM和1 μM的Mlx-8时，其对10 μM卡巴胆碱诱导的总[3H]肌醇磷酸积累的阻断率分别为43.31±8.86%（n=3）和97.42±2.02%（n=3），这表明Mlx-8可抑制海马中与mAChRs激活相关的细胞内信号通路。结论：本研究结果首次证实，眼镜蛇科（Elapidae）条纹珊瑚蛇（Micrurus lemniscatus）毒液来源的Mlx-8毒素可作用于毒蕈碱受体，该毒素是一种兼具磷脂酶A2特性的毒蕈碱配体，能够在大鼠海马组织中与毒蕈碱配体[3H]QNB竞争结合位点。此外，Mlx-8还可阻断毒蕈碱激动剂卡巴胆碱诱导的总[3H]肌醇磷酸积累。因此，Mlx-8有望成为研究毒蕈碱型胆碱能功能的新型药理学工具。