The broad, long term objective of this project is to define the molecular mechanisms by which general anesthetics act. The overall hypothesis to be tested is that general anesthetics alter interactions between membrane proteins and lipids that are critical for maintaining normal protein function. This hypothesis is based on evidence that membrane protein function is sensitive to the lipid environment and requires that anesthetics act on protein conformational transitions that are modulated by lipid-protein interactions.
The specific aims of this project are: (1) to precisely identify the rate constants and agonist affinities governing nicotinic acetylcholine receptor (nAcChoR) desensitization that are sensitive to general anesthetics; (2) to determine whether anesthetic-sensitive rate constants and agonist affinities are modulated by lipid-protein interactions; (3) to identify the lipids that confer anesthetic sensitivity to reconstituted nAcChoRs and to test the hypothesis that anesthetics compete with these lipids for hydrophobic sites on nAcChoRs; and (4) to determine whether nonanesthetic compounds alter nAcChoR desensitization kinetics and to test the hypothesis that anesthetics and nonanesthetic compounds compete for discrete nAcChoR binding sites. The research design is to characterize the actions of general anesthetics on nAcChoRs in native membranes from Torpedo and then to alter lipid- protein interactions by reconstituting nAcChoRs into lipid bilayers whose cholesterol and phosphatidic acid (PA) content varies. The hypothesis that anesthetics compete with lipids for hydrophobic sites on nAcChoRs will be tested by assessing the anesthetic sensitivity of nAcChoRs that have been reconstituted into bilayers whose cholesterol and PA content varies. The hypothesis that anesthetic and nonanesthetic compounds compete for binding sites on the nAcChoR will be tested by determining whether nonanesthetics reduce the potencies with which anesthetics act on nAcChoRs. The method used to characterize nAcChoR conformational transitions will be stopped-flow fluorescence spectroscopy. This technique has a time resolution that is nearly 1000 fold faster than radioligand techniques typically used to characterize nAcChoR desensitization kinetics. The nAcChoR will be used as the protein model because it is the best characterized ligand-gated ion channel, it is sensitive to general anesthetics, and it is only one that can be purified in the quantity and specific activity needed for biophysical studies.
