instnjctions): The overall aim is to gain a better understanding of the manner in which general anesthetics interact with the activated states of the ligand-gated ion channels of the Cys-loop superfamily. The overall hypothesis is that general anesthetics interact with these channels at allosteric sites whose properties vary with the channel's conformational state. A given binding site's size and affinity for anesthetics varies with time as the receptor changes its conformation following addition of agonist. The approach is structured to encourage a tight integration between kinetic and structural approaches with kinetic studies generating hypotheses about the relative affinity of anesthetics for specific transient conformational states and time-resolved freeze quenched photolabeling being used to detemriine the degree of photoincorporation and to locate anesthetic sites.
Aims 1 and 2 probe the mechanisms and binding sites involved in anesthetic enhancement of agonist- induced ion currents.
Aim 1 addresses this issue for the cationic members of the family where only the smallest anesthetics enhance currents. The specific hypothesis to be tested is that the enhancing site increases in size as the receptor passes from the closed to the open and then desensitized states. The kinetics of this are most easily dissected on the slowest member of the superfamily, the human neuronal 5 HT3AR.
Aim 2 will locate the enhancing site in human neuronal GABAARs in collaboration with Project 3 and using photoactivable anesthetics from the etomidate, propofol, barbiturate and alcohol families. The second part of this aim asks whether the site at which etomidate itself activates GABA currents is the same as that which enhances agonist-induced currents.
Aim 3 explores the role of binding sites at the subunit interface vs. those in the ion channel in inhibition and enhancement using probes specifically designed for the task. This work will use the Torpedo acetylcholine receptor because its known structure will facilitate interpretation. In each aim, the receptor chosen is that best suited to answering the question. Photolabels and purified, heterologously expressed receptors will be provided by the Synthetic Chemistry and Protein Production Cores respectively, and sequencing by the Protein Chemistry Core.
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