Effects of Mlx-8, a phospholipase A 2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

• Published in: The journal of venomous animals and toxins including tropical diseases Vol. 26; p. e20190041
• Main Authors: Dos Santos, Roberta Tancredi Francesco, Silva, Marcelo Florencio Passos, Porto, Rafael Marques, Lebrun, Ivo, Gonçalves, Luís Roberto de Camargo, Batista, Isabel de Fátima Correia, Sandoval, Maria Regina Lopes, Abdalla, Fernando Maurício Francis
• Format: Journal Article
• Language: English
• Published: Brazil 2020
• Subjects: Phopholipase A2; Micrurus lemniscatus; Inositol phosphate; Muscarinic receptors; Hippocampus
• ISSN: 1678-9199; 1678-9199

Here, we described the presence of a neurotoxin with phospholipase A activity isolated from venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). The purification, molecular mass determination, partial amino acid sequencing, phospholipase A activity determination, inhibition of the binding of the selective muscarinic ligand [ H]QNB and inhibition of the total [ H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. 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 A enzymatic activity. The pK values were determined for Mlx-8 toxin and the M selective muscarinic antagonist pirenzepine in hippocampus membranes via [ H]QNB competition binding assays. The pK 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 [ H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A . This suggests that the inhibition of the phospholipase A activity of the venom did not alter its ability to bind to displace [ H]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 [ H]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. 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 A characteristics, obtained from the venom of the Elapidae snake , since this toxin was able to compete with muscarinic ligand [ H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [ H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.