Preparation, Pharmacological Property And Interaction Mechanism Of Scorpion Toxins Targeting Sodium Channels

Mammalian sodium channels play pivotal roles in physiological and pathological functions, and the studies on their structure-function relationships progress slowly. Various scorpion toxins specific for sodium channels are important molecular tools in probing channel structure-function relationships. These toxins are polypeptides containing60-70amino acid residues and stabilized by four disulfide bridges. At present, the preparation of scorpion toxins still remains a huge challenge. Because of the tiny amount and complexity of the crude venom, it is highly difficult to obtain more than1mg specific scorpion toxin for in-depth studies. The genetic engineering technique has been used for the preparation of extremely limited scorpion toxins, and some unknown factors influnce the production of more toxin. As a result, it is highly essential to explore the preparation of scorpion toxins by the genetic engineering technique with low cost and easy application, which will accelerate understanding of toxin structure-function relationships, and promote their biological and medical application.In this thesis, we firstly studied the expression strategy and pharmacological properties of several scorpion toxins. We screened candidate scorpion toxins from the scorpion Buthus martensii Karsch and Lychas mucronatus, and expressed the toxins BmaTX47, BmaTX14, BmKIM, Lm3272and Lm4404by using expression vectors pET-14b and pET-28a respectively through the in vitro folding strategy. The toxins BmaTX47and BmaTX14were found to be successfully expressed with these expression vectors; The toxin BmKIM was only obtained with the pET-14b vector; The putative toxins Lm3272and Lm4404were not prepared using both vectors. Pharmacological experiments showed that the expression vectors strongly affected the activities of the recombinant toxins. For example, the10?M rBmaTX47from the pET-28a vector inhibited the fast inactivation of rNav1.2and hNav1.5channel currents with the I5ms/Ipeak values of44.12±3.17%and65.34±3.86%, respectively. Such potency were much better than those values of11.33±1.46%and5.24±2.38%for these two sodium channels by10?M rBmaTX47from the pET-14b vector. However, the recombinant toxin rBm?TX14from expression vectors pET-14b and pET-28a showed less significant difference in their efficacies towards mNav1.4channels with the EC50values of32.68±6.51nM and82.3±15.7nM, respectively. Besides, the toxin BmKIM from pET-14b slightly delayed the fast inactivation of rNav1.2current rather than mNav1.4and hNav1.5at the concentration of1?M. These results indicated that more scorpion toxins should be prepared using different expression strategies, and the underlying factors affecting the pharmacological properties of scorpion toxins could be explored, which might lay the foundation for further basic and applied studies of scorpion toxins in the future.Then, we focused on critical residues of sodium channel mNav1.4responsbile for scorpion toxin Bm?TX14recognition. Using the mutageneis technique, we scanned the residues in DIV/S1-S2and DIV/S3-S4loops of mNav1.4channel. In the DIV/S1-S2loop of mNav1.4, pharmacological experiments showed that single mutants E1367A, V1376A, D1377S and N1381W significantly decreased the channel potencies towards Bm?TX14by71.12-,67.53-,72.47-and68.31-folds, respectively. In the DIV/S3-S4loop, pharmacological experiments indicated that mutants G1424F, D1429A, D1429E, Q1432D and K1433D reduced the potency of Bm?TX14by85.3-,1259.5-,421.8-,40.19-and480.2-folds, respectively. These results highlighted the important roles of DIV/S1-S2and DIV/S3-S4loops in the sodium channel for Bm?TX14binding. Considering the minor effect of toxin Bm?TX14on sodium channel rNav1.2(EC50=54193±1300nM), we further exchanged the extracellular loops of DIV between sodium channels rNav1.2and mNav1.4. It was found that DIV/S3-S4on sodium channels was the pivotal domain for Bm?TX14recognition, while the DIV/S1-S2loop was also involved in the interaction between Bm?TX14and sodium channels. Based on the indentification of functional residues in sodiucm channel Nav1.4for Bm?TX14binding, molecular dynamic simulations were used to constructe toxin-channel complex model. The structural model showed that toxin Bm?TX14was docked to the extracellular side of Domain ? in sodium channel Nav1.4, with the residues8-18wedged into the binding pocket. Several hydrophobic clusters and salt bridges were observed between the binding interfaces of Bm?TX14 and Nav1.4, which possibly stabilized the toxin-channel complexes. These results elucidated the binding pattern between a-like scorpion toxins and sodium channels.Finally, we tried to figure out the molecular basis of scorpion toxins on their preference for sodium channel subtypes. The a-like toxin BmaTX14selectively inhibits the inactivation of Nav1.4rather than Nav1.2channels, while the classical a-toxin Lqh2is potent for both Nav1.2and Nav1.4channels. We focused on the significantly different functional residues on the bioactive surfaces on toxins BmaTX14and Lqh2, including positions7-10,15-21,39-41and54-64(C-terminal). Through interconverting the different functional domains between the two toxins, the analysis of the efficacy and selectivity of the toxin mutants was used to identify the essential domain affecting toxin preference for sodium channel subtypes. When positions15-21residues were exchanged between BmaTX14and Lqh2, it was found that the toxins preferences for Nav1.2and Nav1.4channels have been reversed for both BmaTX14and Lqh2mutants. These results highlighted the critical role of position15-21in toxin potency for sodium channel subtypes. In addition, it was found that other different domain from both NC-domain and Core-domain also affected the efficacy and selectivity of scorpion toxins. These findings would prompt the design of a-scorpion toxins as specific molecular tools and drugs in the future.In conclusion, we expressed several scorpion toxins specific for sodium channels, and revealed the important role of expression vectors pET-14b and pET-28a in the toxin pharmacological properties. Based on the potent toxin Bm?TX14, we studied the functional residues and their distribution in sodium channel Nav1.4responsible for BmaTX14binding. We also studied the molecular basis of scorpion toxins on their preference for sodium channel subtypes. These results laid the foundation of standard expression technique for further studies on the structure-function relationships of scorpion toxins, and promoted the design and application of scorpion toxins as therapeutics and potent molecular tools.