The goal of this research proposal is to examine how the number, gating properties, and conductance of synaptic receptors may be altered by changes in a cell's lipid membrane. The project will involve manipulations of the plasma membrane lipid composition of tissue-cultured muscle cells and the measurement of subsequent changes in the number of nicotinic acetylcholine receptors, as reflected by alpha bungarotoxin binding, and in the activity of these receptors, as monitored by electrophysiological recordings with the patch-clamp technique. The ability to record the activity of single, nicotinic receptors with the patch-clamp technique has revealed a variety of open and closed states in this prototypic synaptic receptor. These studies are designed to detect the extent to which cell-mediated changes in membrane lipids could affect receptor transitions between these various open and closed states as well as changes in the number of receptors in the plasma membrane. Cells from the BC3H-1 muscle line will be cultured under conditions which will alter the plasma membrane phospholipid fatty acid composition, membrane content of cholesterol, or content of phospholipid head groups. Following analysis of the induced lipid changes, the number of nicotinic receptors in the plasma membrane will be assayed with 125I-alpha-bungarotoxin binding studies and the behavior of nicotinic receptors in these cells will be monitored by patch-clamp electrophysiological recording. The relative occurrence of the different conducting states of the receptor, the frequency of transitions between these states, and the dependence of receptor channel opening upon agonist concentration will be measured as functions of membrane composition. The effectiveness of various pharmacological agents at different portions of the receptor will be used to probe for changes in the receptors that are not readily revealed by their electrical responses. These agents will include channel blocking drugs for studies of changes in the open receptor channel and chemically different agonists and competitive antagonists for studies of changes in agonist binding.
