| This thesis compiles experimental results concerning gating mechanisms of a small proton gated potassium channel from Streptomyces lividans (KcsA). The cloning of KcsA in 1995 represents the starting point of our scientific work at the University of Virginia in the laboratory of Dr. Eduardo Perozo.; After KcsA cloning, our laboratory become one of the few able to express milligrams quantities of this protein, which by the way was extremely stable, even at very high temperature. Because KcsA robust expression in E. coli; this protein become an excellent biochemical preparation. Chapter 7 of this thesis explains in detail how KcsA can be gated, and become the first scientific report explaining KcsA proton dependent activation. A combination of site directed mutagenesis, site directed spin labeled, Electron Paramagnetic Resonance spectroscopy (EPR), bulk functional assays and single channel characterization, allows to present the topological arrangement of this potassium channel in a lipid membrane and the conformational changes that take place upon activation gating. Chapter 8 and 9 of this thesis explain in chronological order how these experimental findings were achieved. Simultaneously, Declan Doyle and Roderick MacKinnon produced the landmark paper of the crystallographic solution of KcsA structure, which elegantly set the molecular basis of ion permeation and selectivity in a potassium channel. This work represents one of the most important contributions to make Roderick MacKinnon the recipient of the Nobel Prize award in Chemistry. Chapter 10 of this thesis is a compendium of experimental results that explain how structural elements along the permeation pathway are coupled and ultimately control KcsA gating behavior.; The most obscure aspect of KcsA function is exactly the one that makes it so accessible to Electrophysiologist, its pH sensitivity. In chapter 11, I present a systematic characterization of KcsA pH dependence, which ultimately lead to propose a structural model that explain what are the molecular basis for pH dependence. The last chapter of this thesis is a short demonstration of the putative role played by KcsA-like channels in bacterial physiology and supports the role of protons as real activators of KcsA-like proteins. |
