Investigations of ion channel structure-function relationships using molecular modeling and experimental biochemistry

Ion channels are integral membrane proteins found in all cells that mediate the selective passage of specific ions or molecules across a cell membrane. These channels are important in a diverse range of physiological processes, including signal transmission in the nervous system, sensory perception, and regulation of vital systems, such as circulation. This thesis discusses the use of computational chemistry methods, such as molecular dynamics (MD) and ab initio calculations, and experimental biochemical techniques, such as site-directed mutagenesis, in vivo bacterial assays, chemical cross-linking, and circular dichroism spectroscopy, in tandem to elucidate ion channel structure-function relationships. This research was catalyzed by the solving of atomic resolution crystal structures of the mechanosensitive channels of large and small conductance (MscL and MscS) by the Rees group. Although interesting themselves, these bacterial channels also provide good model systems for considering more complex eukaryotic channels. MscL is an ion channel gated only by membrane tension. Initial studies of MscL verified the relevance of the crystal structure conformation under physiological conditions and compared different MscL homologues. Other work began to elucidate potentially unique structural and functional roles of the M. tuberculosis MscL C-terminal helical bundle. As well, interactions between the MscL channel protein and surrounding lipid and the potential relevance of helical kinking in MscL gating pathways were investigated. MscS is also gated by membrane tension, but its gating can be modulated by changes in transmembrane potential. Thus, studies on MscS began to identify the specific amino acid residues that are responsible for giving the channel its voltage sensitivity. Finally, computations predicting the conformation of nicotine in different solvent environments are discussed. Nicotine is a small molecule ligand that binds to and gates nicotinic acetylcholine receptors, and a thorough understanding of nicotine structure could aid efforts to elucidate receptor structure-function relationships and design new pharmaceuticals.