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.
Showing the most recent 10 out of 21 publications
