Studies On The Interaction Mechanism Of ?/?1 Subunits And The Extracellular Sturcture Of ?2 Subunits Of BK Channels

Ion channels are macromolecular pores in cell membrain, which are the fast channels of water-soluble matters including charged ions going into and out of the cell. BK channels, large conductance voltage-dependent and calcium- activated potassium channels,play a critical role in the life activities associated with different auxiliary subunist,participating in regulating electrical signaling in nerve, muscle, and synapse. Ion channels are also closely related to a variety of genetic diseases. Intensive studies of BK channel gating mechanism and dynamic characteristics, is of far-reaching practical and instructive significance for revealing the mechanism of diseases and looking for the therapeutic method.The reserch work of this article mainly consists of two parts:The first part is focused on the study of Calcium and Magnesium regulation of BK channels with ?1 subunits, by combination of the patch clamp technique, molecular biology and homology modeling. The mechanism and interacting sites are also investigated. BK channels associated with ?1 subunits exhibits an enhanced calcium sensitivity and slow deactivation kinetics. BK(?1) mainly expresses in smooth muscle cells and plays an important role in the proper regulation of smooth muscle tone . The main results and conclutions of this section are as follows:(1) We identified an enhancing (E) site (mSlo1(K392,R393)::?1(E13,T14)),which increases the calcium sensitivity of BK channel.(2) We further found a hydrophobic (H) site (mSlo1(L906,L908)::?1(L5,V6,M7)), passing the physical force from the Ca2+ bowl onto the E site and S6-Clinker.(3) Based on the above experimental results, a comprehensive structural model of the BK(?1) complex was reconstructed.The second part is concentrated on the identification of the pairing mode of the cysteine residues in the extracellular loop of BK ?2 subunits, ultlizing the patch clamp technique together with molecular biology and homology modeling. BK(?2) channels mainly expressed in chromaffin and hippocampus cells, and behaved rapid inactivation and slow deactivation. The extraceelular loop of ?2 subunits played an important role in regulating the outward rectification and toxin sensitivity of BK channels, which contributed to the neural signal transduction. The main results and conclutions of this section are as follows:(1) We successfully identified three pairs of disulfide bonds formed in the external loop of ?2 subunit, and the rest two cystine residues remain solitary but located much colser to the channel pore.(2) Based on the information provided by electrophysiological and biochemical experiments, we also built a structure of ?2 subunit and then composed a complex of mSlo1 and ?2. In this study, we have provided not only a novel method of recognizing the pairing mode of cysteines, but also a basic structural comformation of external loop of ?2,which should be very helpful in understanding the crystal structure of ?2 in the future.In conclusion, this article investigates the foundamental properties of BK channels,and provides a functional and structural understanding of BK with different ? subunits.