The overall goal of this project is to determine the role that protein-lipid interactions play in the functioning of the nicotinic acetylcholine receptor (AChR), which is the best-studied example of a ligand-gated ion channel. A number of studies using both native membranes and reconstituted systems have demonstrated that bilayer composition has effects on AChR structure and function, and protein labeling experiments have identified several regions of the receptor that are at the protein-lipid interface. We will examine the role(s) that lipid- exposed regions of the AChR play in receptor gating by approaching this problem from both the direction of the membrane and the receptor protein by altering both parts independently. In this proposal we will use a combination of electrophysiology, fluorescence spectroscopy, and molecular biological techniques to examine the influence that membrane composition and specific sites of AChR-lipid contact play in the gating properties of the AChR. We will use cyclodextrin-mediated transfer of cholesterol either into or out of the membrane to increase or decrease membrane cholesterol content of transfected cells expressing AChRs, and then analyze the kinetics of ACh-induced channel opening as a function of membrane cholesterol content. In parallel with these electrophysiological studies, we will carry out fluorescence spectroscopy studies using fluorescence anisotropy of diphenylhexatriene and general polarization of laurdan to examine the physical state of the bilayer of the transfected cells in order to correlate changes in AChR gating behavior with alterations in membrane properties. We will also carry out a series of site-directed mutagenesis studies targeted at residues known to be at the protein-lipid interface in order to examine the types of interactions that these residues make with either the lipid environment or other regions of the receptor in order examine the role that these residues may play in AChR gating. These studies will then be expanded to include examination of the effects of altering membrane properties through changes in membrane cholesterol content on the altered properties of the mutant receptors. It is expected that these studies will provide insight into how the membrane environment influences AChR function, and, by inference, that of the other members of the ligand-gated ion channel family.
