The Expression Optimization For The Martentoxin And The Struture Simulation Of BK Channel Pore

Large conductance calcium activated potassium channels(BK) are voltage and calcium ions double gated, which are widely distributed in excitable and nonexcitable cells. They are key participants in a variety of physiological functions, such as regulating cell excitability, controlling cell proliferation, migration and the osmotic pressure. A 37-residue short-chain peptide, named as martentoxin, arising from the venom of the East-Asian scorpion(Buthus martensi Karsch) has been investigated with a comparatively higher preference for BK channels over other voltage-gated potassium(Kv) channels. Up to now, since the specific drug tool probing for clarifying structure-function of BK channel subtypes and related pathology remain scarce, it is of importance to illuminate the underlying mechanism of molecular interaction between martentoxin and BK channels. This will help us analyze the structure, function and molecular mechanism in related pathology of BK channel furtherly.This work mainly adopts the method of molecular biology and computer simulation. First, compare different expression vectors and competent cells to choose optimal carrier p GEX-4T-3 and competent cell Rosetta eventually, with 16 degrees induction temperature, 24 h induction time and 0.5m M inducer dosage. According to pharmacological function identification, the recombinant Martentoxin can suppress the current of BK channel effectively. This was conformity with the natural pharmacological properties of Martentoxin, verifying its pharmacological combination activity. Second, use potassium channels Kcs A from prokaryotes Streptomyces lividans as molecular skeleton to construct Kcs A-BK membrane protein chimera which containing the ions through hole zone microdomain of BK. Next select suitable carrier p QE30 and competent cell M15 depending on the E. coli expression system to achieve the low cost efficient expression of Kcs A-BK. And we use biological macromolecular interaction platform Bia Core T200 combined with molecular technology to explore the interaction of martentoxin with the microdomain of BK channel target receptor site, proving that the chimeric Kcs A-BK membrane protein expressed in vitro can pharmacologically combine with martentoxin specifically. Moreover, discovery studio was utilized to simulate BK pore’s structure. With bioinformatical methods, the possible recognition sites of BK channel-specific ligands will be predicted and verified for the optimized binding, which could help to forward the ideal channel-ligand interaction model and associated envionmental factors.This work lays solid methodological foundation and theoretical expanding space for exploring new target for drugs subsequently as well as designing related channel disease treatment drugs.