General anesthetics are widely used during surgery and other clinical procedures, yet they are extremely dangerous and their mechanisms remain poorly understood. Understanding their actions at the molecular level will enable development of new drugs with more therapeutic specificity. The overall aim of this project is to define the mechanisms of potent general anesthetics at known therapeutic targets: synaptic and extrasynaptic GABAA receptors. Photolabeling, structural homology modeling, mutant studies, and mechanistic analysis have established structural and functional models for etomidate actions at synaptic aiP2Y2L receptors, providing a new paradigm for other potent anesthetics such as propofol and barbiturates, and extra-synaptic GABAARS containing different subunits. Our working hypothesis is that at synaptic cci(32/372 and extrasynaptic OAPSS GABAA receptors, different potent anesthetics bind to distinct sub-regions of intra-membrane pockets formed at a-p subunit interfaces, with each subunit contributing a channel-lining transmembrane M2 domain and one other: a-M1 and P-M3. Anesthetic binding facilitates rearrangement of these pockets, stabilizing open channel states, thereby reducing neuronal activity. We propose to electrophysiologically study expressed GABAARS of defined subunit composition, developing a detailed mechanism of anesthetic actions at the macrocurrent level and determining how structural changes in the putative drug pockets affect anesthetic interactions and channel gating.
Specific Aim 1 is to investigate anesthetic-photolabeled amino acids in aip2/3y2L (synaptic) GABAARS and their roles in channel gating and the affinity/efficacy of etomidate, propofol, and pentobarbital. Mutagenesis, including cysteine mutagenesis and state-dependent modification, together with electrophysiological analysis based on an equilibrium co? agonist gating model will be used.
Specific Aim 2 is to build an allosteric-kinetic model for etomidate modulation of aiP2/3'y2L GABAARS and to develop allosteric co-agonist models for propofol and pentobarbital. Macrocurrent kinetic electrophysiology will be performed using an """"""""artificial synapse"""""""" for rapid application of GABA and anesthetics, and kinetic models will be assessed for their ability to quantitatively account for the data.
Specific Aim 3 is to extend the above approaches to a4p35 (extrasynaptic) GABAARS.
Specific Aim 4 is to collaborate with other PPG projects by assessing new photolabel anesthetics and guiding time-resolved photolabeling experiments.
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