This proposal is Project III of a new Program Project Grant entitled """"""""General Anesthetic Sites on Ligand gated Ion Channels."""""""" The long term objective of this project is to define how and where general anesthetic drugs modulate the function of neurotransmitter-gated ion channels in the brain. This information is essential for designing safer general anesthetics. The working hypothesis is that anesthetics act by binding to hydrophobic sites on the superfamily of homologous """"""""cys-loop"""""""" ligand-gated ion channels that includes nicotinic acetylcholine receptors (nAChRs) and gamma-aminobutyric acid receptors (GABAAR). This proposal focuses on both nAChRs and GABAARs, because actions of anesthetics in nAChRs and GABAARs show parallels; both inhibition and potentiation are observed, depending on the receptor state and the anesthetic drug. The nAChR is an experimental model where structure, function, and mechanisms of anesthetic actions are better defined than those for other ion channels. GABAARs are likely to be clinically important targets for general anesthetics in the brain.
Specific Aim 1 is to define which kinetic states (resting, open, or desensitized) of nAChR and GABAAR are affected by anesthetics. Recombinant, expressed nAChR and GABAAR receptors and rapid-perfusion electrophysiology techniques that mimic natural synapses will be used to study the actions of both inhibitors and potentiators.
Specific Aim 2 is to determine whether regions of the nAChR protein that are photo-labeled by anesthetics (PPG projects I and II) form binding sites where anesthetics cause their various actions. One discrete site, where anesthetics block the open nAChR pore, has been identified and partially mapped. By mutating other anesthetic binding regions in recombinant nAChRs, specific models detailing where anesthetics act will be tested.
Specific Aim 3 is to determine whether GABAAR inhibition and potentiation sites are formed by regions homologous to those where anesthetics bind to the nAChR.
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