The nicotinic acetylcholine receptor channel (nAChRC) is the best understood ion channel, it transduces the chemical signal at the neuromuscular junction and is the target of pathogenic antibodies in myasthenia gravis. The goal of this proposal is to better understand how the channel protein structure produces its functional properties, specifically, how electrical charge on the surface of the protein influences ion transport. A series of receptor channels with single charged amino acid mutations will be created by site-directed mutagenesis using a uracil-enriched template cDNA. Channel protein expression in Xenopus oocytes and single channel recording under conditions of varied ionic strength and permeant ion concentration will be used to determine the conductance of the channel. The conductance and the shape of the current- voltage relations of the mutant receptor channels will be compared to the wild type receptor. These data will be interpreted in terms of a computational model of the electric potential profile of the nAChRC. From this analysis the relative electrical distances of the point charges to the pore mouth will be calculated. Examination of the sidedness of the effect of changes to the pore mouth will be calculated. Examination of the sidedness of the effect of changes in ionic strength and the rectification of the current-voltage relation will allow the localization of the charge change to the cytoplasmic or extracellular side of the pore complementary to what is currently known about the transmembrane topology from protein chemistry and antibody analysis. The candidate, through work on this project, hopes to refine his skills in ion channel expression and single channel recording, and acquire new skills in DNA cloning and site-directed mutagenesis. He will ultimately apply these techniques to the study of voltage-dependent ion channels from cardiac tissue, specifically the sodium channel from ventricular muscle. He hopes to study questions related to the molecular nature of the local anesthetic antiarrhythmic drug binding site in this channel.
