The role of nicotinic acetylcholine receptors (nAcChoR) in some manifestations of alcohol abuse is being increasingly recognized. Our overall hypothesis is that ethanol's action on postsynaptic membranes is a complex function both of ethanol's concentration and of the degree, rate and duration of stimulation by the agonist. Our overall aim is to determine which of the discrete conformational changes mediating the action and regulation of the nAcChoR is modulated by ethanol, and then to determine the molecular mechanisms underlying its actions. We will use nAcChoRs isolated from Torpedo electroplaques which are 10,000-fold more abundant than any other postsynaptic receptor, the Torpedo nAcChoR's initial channel opening response to acetylcholine (AcCho) is enhanced by ethanol - an action unlike that of other anesthetics. A striking new finding is that ethanol enhances fast desensitization at inebriating concentrations (>10mM). Like other anesthetics, ethanol also accelerates the slow desensitization of nAcChoR during prolonged exposure to AcCho.
Our first aim i s to determine the exact points in the kinetic pathway (see above) where ethanol modulates acetylcholine's action. The partial agonist, nicotine, interacts with ethanol in an unusual way and will also be studied in detail. Agonist-induced cation flux will be measured over times as short as 1 msec. using quenched flow techniques.
Our second aim i s to determine whether ethanol's actions are mediated by a binding site or a nonspecific (lipid) mechanism using the following criteria: (i) the dependence of rate constants on ethanol & agonist concentration (is it saturable or linear?); (ii) pressure reversal of ethanol's actions, (iii) structure-activity relationships of related alcohols (including enantiomers and conformationally restricted alcohols).
Our third aim i s to probe the state of the nAcChoR's membranes's micro-environment, both in the lipid bilayer and adjacent to the protein (""""""""boundary or annular lipid""""""""), as a function of ethanol concentration and of pressure. Does ethanol compete statistically for the lipid-protein interface or actually weaken the interaction between lipid and protein? We will employ various spin labeled lipids. Changes in fraction of spin label in bilayer and annular environments will be determined by spectral subtraction and changes in exchange rate by spectral simulation. Cholesterol also occupies nonannular sites on the protein which can be examined using fluorescence quenching. Finally, within the limitations of the available preparations, we will use pharmacological criteria to classify the action of ethanol on receptors in the same superfamily (Brain AcChoR, GABAR) - are there common mechanisms? A complete knowledge of the kinetics of the AcChoR will enable ethanol's actions on it to be accurately modeled, while understanding mechanisms will allow new therapeutic interventions to be designed.
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