Modulation Mechanism Of Auxiliary Subunits To Potassium Channel Using Solution NMR And Electrophysiological Methods

Ion channels exist in single-celled and multicellular organisms, either animals or plants, with a great diversity of types. Up to now, the investigation of ion channels relies mainly on the ion current of ion channel recorded by patch clamp techniques, from which the functional and structural characters can be further studied by the aids of other techniques. In this thesis, we will discuss the structural and functional study of the potassium ion channel auxiliary subunits family, using solution Nuclear Magnetic Resonance (NMR) and electrophysiological techniques. There are four chapters is this thesis.Chapter1is a brief review of ion channels and patch-clamp technique. First, there is a brief introduction of ion channels. Then the potassium channel family, inclouding the pore-forming subunits and auxiliary subunits were introduced. Finally, We describe the principles of electrophysiological techniques, especially patch-clamp technique.Chapter2is. Brief descriptions of the fluorescent protein technology and solution NMR are also introduced.In chapter2, we investigate the structure and dynamics of potassium channel regulatory protein KCNE2. This work was carried out by ChaoHua Lai (graduated from our laboratory). In order to maintain the integrity of this thesis, this section is still included in this paper.An in-depth study on the modulation mechanism of KCNE2to KCNQ1was carried out. The conductance and dynamics of KCNQ1could be modulated by different single-transmembrane auxiliary proteins, such as KCNE1and KCNE2. Despite of a relatively high secondary structure homology, the three-dimensional structure differs between KCNE1and KCNE2, which may be responsible for their different modulation function on KCNQ1. Here, the comparative study of different domains of KCNE1and KCNE2were carried out using combinational methods of electrophysiology, immunofluorescence, solution NMR and backbone flexibility analysis.Chapter3describes the structure and function of another potassium channel auxiliary subunit MPS1. MPS1is also a member of KCNE family, first discovered in nematode C. elegans. Previous studies were reported that the intracellular carboxyl terminus of MPS1has serine/threonine kinase activity. However, we found that the intracellular carboxyl terminus of MPS1adopts a high flexible random coil conformation using solution NMR. This result strongly indicated that the MPS-1protein can not form the stable three dimensional folding for a serine/threonine kinase, which was confirmed by functional assays.In chapter4, first-step functional study on BK, another important potassium channel, is discussed. The a subunit and its modulatory subunits, β1and β2, were transfected in mammalian cell line CHO-k1. The current was recording using whole cell patch.