Structural studies of BK ion channel in a lipid environment

Thesis (Ph.D.)--University of Washington, 2018Integral membrane proteins carry out a variety of functions, including cross-membrane transportation, signal transduction, and cellular metabolic regulation. For human beings, around 18.5% of the dry weight of cells are membrane proteins, which are vital for the cells. However, according to a membrane protein database, only a small number of unique membrane protein structures had been identified before the start of my study. This is mainly due to the technical challenges associated with expressing membrane proteins in large quantities, solubilizing them in appropriate detergents and crystallizing them for X-ray crystallography. Cryo-Electron Microscopy has emerged as a powerful method for structural studies of membrane proteins. A central goal of my graduate research is to determine the structure of the human large-conductance voltage- and calcium -activated potassium (hBK) channel in a lipid environment using cryo-EM. To restore the asymmetric lipid membrane environment of membrane proteins, a method called “random spherically constrained” (RSC) single-particle cryo-EM was developed. Voltage-gated ion channels sense changes in membrane potential and undergo conformational changes that regulate ion flux across membranes in excitable cells. Due to the lack of methods to apply transmembrane potentials for structural studies, the mechanism under which voltage-gated ion channels respond to the voltage change across the membrane remains enigmatic. The liposome system I am using can establish a native lipid environment for membrane proteins and make it possible to apply transmembrane potentials to trap voltage-gated ion channels in desired functional states for structural analysis. I have determined the structure of hBK at an intermediate state, at 3.5 Å resolution with no membrane potential and in the absence of calcium. Overall, my graduate research has improved our understanding of the human BK channels and optimized the sample preparation procedures for RSC cryo-EM method. Furthermore, this work established the methodology of controlling transmembrane voltage for cryo-EM single particle analysis, which may provide insights into other voltage-gated channels