The molecular mechanism of action of general anesthetics in the central nervous system is hard to define, but anesthetics are believed to act by preventing synaptic transmission. Anesthetics do act on peripheral synapses and we propose to continue to study their effects at the nicotinic receptor membrane of Torpedo, which offers the opportunity to relate changes in the receptor's function to changes in its membrane structure. All general anesthetics examined enhance slow desensitization of the receptor reversibly with relative potencies which correlate closely with those for disordering the lipid bilayer of this acetylcholine receptor-rich membrane. The correlation might easily arise by chance. To test it further, a number of incisive pharmacological tools will be employed. (1) does pressure reverse anesthetic-induced desensitization and lipid disorder in parallel. (2) In many homologous series (alcohols, alkanes) anesthetic potency initially rises as the chain length in increased but then, paradoxically, after the addition of one more methylene group all activity is lost (the cut-off effect). Does the chain length at which cut-off occurs correspond to that where disordering ceases? (3) Do cis- and trans-alkenols show stereoselectivity and (4) is the structural specificity of steroid anesthetic action the same for desensitization and lipid disordering? The mechanisms underlying desensitization will then be probed by measuring the kinetics of binding of a fluorescent cholinergic ligand on the millisecond time scale using the stopped-flow technique. Which of the various discrete conformational changes leading to the desensitized receptor state is affected by anesthetics? Does the established overall antagonism of anesthetic effects by pressure occur at one kinetic step or does it represent an indirect compensation? Spin- labeled lipids will be used to test the hypotheses that general anesthetics and pressure act in opposing directions by perturbing: (1) the bulk membrane lipids, and (2) lipid-protein interactions. Do the anesthetic concentrations and pressures causing these structural perturbations correlate with those associated with the functional changes? Acetylcholine receptors also undergo fast desensitization. The pharmacology of this transition is unknown. It will be explored using stopped flow and quenched flux measurements. General anesthetics also inhibit cation flux through the receptor's ion channel but with a more specific pharmacology than desensitization. This pharmacology will be further explored, and then pulsed quenched flux techniques will be used to define the underlying kinetics of anesthetic and hyperbaric action.
NIH ExPorter
NIH ExPorter