Michael M. White, PhD - US grants

This proposal is for the continuation of our studies using the combination of molecular biology, biochemistry, and electrophysiology to understand how ion channel structure defines function. We will focus on two different members of the ligand-gated ion channel family: the muscle- type nicotinic acetylcholine receptor (AChR), which is the best- characterized member of the family, and the serotonin type 3 receptor (5HT3R), which is the least-characterized member. We anticipate that many of the features that underlie ion channel function represent variations on a common structural theme, so that information obtained from the AChR represents a conceptual framework from which to approach 5HT3R structure- function studies, and vice versa. One goal of this project is to examine to what degree this commonly-held notion of cross-receptor applicability holds true. Our approach is to create a series of predetermined single (or double) amino acid substitution mutants using site-directed mutagenesis, and then to compare the properties of the mutant receptors to those of the wild- type using electrophysiological and ligand-binding assays after expression in mammalian cells. We will focus on the ligand-binding domains of both the AChR and 5HT3R, and will employ a number of different agonists and antagonists of varying structure to determine particular points of interaction between the ligand and its binding site. In addition, we will investigate the role of residues within the pore itself in the conformational changes that are involved in channel opening and desensitization as a first step in understanding how the binding of a small molecule to the receptor results in channel opening. Finally, we will determine the structure of the ion channel pore of the 5HT3R through the introduction of a series of channel-blocking Ni++-binding sites throughout the length of the channel. From the periodicity (in terms of amino acid position) of the introduction of a binding site the secondary structure of the pore can be determined, while the voltage dependence of the Ni++ block will allow the mapping of the transmembrane electric field onto the physical length of the pore. It is expected that these studies will validate the notion that all members of the ligand-gated ion channel gene family share common structural features, and that many receptor-specific features represent variations on a common structural theme. This may in turn provide some insight into the molecular basis of normal and abnormal cellular activity in a wide variety of excitable cells.

This project is a continuation of studies using a combination of molecular biology and electrophysiology in order to understand how ion channel structure determines function. It will focus on two different ion channels, the nicotinic acetylcholine receptor (AChR), which is the "prototypical" ligand-gated channel, and the Shaker (SH) K+ channel of Drosophila, which is likely to serve a similar role for voltage gated channels. In vitro transcription of cloned cDNAs for the various channel subunits is used to produce large quantities of subunit-specific mRNAs, and the appropriate mixture of RNAs is injected into Xenopus oocytes, which then produce functional channels coded for by those RNAs. Factors that govern channel assembly such as transcript availability and subunit RNA stoichiometry can be manipulated by varying the composition of the RNA mixture that is injected into the oocyte. Site-directed mutagenesis can be used to introduce specific, predefined amino acid changes in the channel, and the structural domains involved in various functions can be mapped out through comparison of the electrophysiological and biochemical properties of the mutant channel with the wild-type. Regions of particular interest in the AChR are the putative ACh-binding domain on the alpha subunit, the sites of phosphorylation by second messenger-activated protein kinases (which are thought to influence the rate of receptor desensitization), and the sites of N-linked protein glycosylation (which may influence receptor assembly through effects on protein targeting and subunit stability, as well as receptor function). The experiments on Sh K+ channels deal with the subunit composition of the channel (monomer vs multimer; homo- vs hetero- oligomer). In addition, site-directed mutagenesis will be used to alter the charge distribution located at the putative "mouth" of the channel to determine the environment that a K+ ion encounters as it enters and exits the channel. In addition, we will alter the charge distribution of the putative voltage sensor of the Sh channel to study the nature of the voltage-dependent gating.. It is expected that these studies on representatives of both classes of ion channels will help us to map out the structural features of ion channels involved in gating, ion channels will help us to map out the structural features of ion channels involved in gating, ion transport, and assembly. It is further expected that some of the structural features relevant to the assembly and function of these two channels will be common to other ion channels, and thus provide insight on the molecular basis of normal and abnormal cellular electrical activity.

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.