The molecular mechanisms of ethanol's actions have proved difficult to elucidate. Some workers evoke direct ethanol-protein interactions, others perturbations of lipid or lipid-protein interactions. Without a resolution of this confusion, the origin of phenomena such as tolerance cannot be understood. Ethanol exerts similar actions on several members of the ligand-gated superfamily ion channels that includes the GABA, 5HT/3, NMDA and nAcCho receptors. One of these receptors provides the best opportunity to solve the mechanisms of ethanol's action because it is 10,000-fold more abundant than the others. This proposal focuses on the nAcChoR (nicotinic acetylcholine receptor), whose role in some manifestations of alcohol abuse is being increasingly recognized. We will use nAcChoRs isolated from Torpedo electroplax which are homologous with mammalian receptors. The overall hypothesis is that ethanol acts on both the protein and the lipid of this receptor but that each action may cause a different functional effect. It is therefore necessary to dissect each action of ethanol, finding the actual rate constants upon which it acts and determining their dependence on ethanol concentration in order to elucidate each mechanism. Our work so far indicates that the two major actions of ethanol are to stabilize the open channel state and enhance desensitization; actions that are apparent at physiological concentrations.
Our first aim i s to characterize the effects of ethanol and temperature on the channel opening and closing rate constants and on fast desensitization using recently developed techniques of superior time resolution.
Our second aim i s to test the hypothesis that ethanol's acute actions and the development of tolerance depend on the composition of the surrounding lipid bilayer. A striking new finding is that ethanol's action on agonist-induced conformational changes is abolished when it is reconstituted into phosphatidylcholine alone, whereas it is present when reconstituted into phosphatidylcholine; phosphatidic acid: cholesterol. The relation between lipid composition and sensitivity to ethanol will be systematically investigated using fluorescent ligands in a stopped flow fluorimeter with special reference to lipids thought to be involved in membrane tolerance.
Our third aim i s to correlate ethanol-induced perturbations in protein and lipid structure, determined using spectroscopic probes covalently attached to lipids or to the nAcChoR, with their functional effects. Our current work leads us to test the hypothesis is that ethanol selectively perturbs slow, but not fast, acyl chain motions near the lipid-protein-aqueous interface and that the nAcChoR's structure is perturbed in the bilayer region but not further away. A second hypothesis is that ethanol interacts with discrete cholesterol sites on the nAcChoR. A complete knowledge of the kinetics of the AcChoR will enable ethanol's actions on it to be accurately modeled, while understanding underlying molecular mechanisms will allow new therapeutic interventions to be designed.
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