The broad. Iong-term objective of this project is to define the molecular mechanisms by which general anesthetics act on protein targets in the CNS and periphery. This will guide the development of new anesthetic compounds possessing fewer side effects. The overall aim is to disentangle the effects of general anesthetics on agonist binding, channel gating kinetics, and agonist-induced desensitization in the best-characterized model ligand-gated ion channel (LGIC), the Torpedo nicotinic acetyicholine receptor (nAcChoR), and to identify the physicochemical features of anesthetics that govern their action on each kinetic step. The overall hypothesis is that general anesthetics act on the nAcChoR in a structurally specific manner because anesthetic binding affinity is strongly influenced by attractive electrostatic and repulsive steric interactions between anesthetics and their protein binding sites.
The specific aims are:
Aim 1 : (1) to test the hypothesis that electrostatic (dipolar, quadrupolar, and/or hydrogen bonding) interactions between general anesthetics and the nAcChoR enhance binding to functionally important sites on this receptor and (2) to identify the kinetic step(s) leading to nAcChoR channel opening that are altered by general anesthetics to determine whether an anesthetic's molecular volume or chemical class governs its action.
Aim 2 : (1) to test the hypothesis that small general anesthetics increase nAcChoR's rate constant for desensitization by binding to a protein binding site that sterically limits the binding of large anesthetics and (2) to test the hypothesis that general anesthetics stabilize the open channel state and increase the rate constant for desensitization by binding to the same small receptor binding site. The proposed studies will lead to a better understanding of the fundamental interactions between anesthetics and their targets in the CNS and periphery. The nAcChoR was chosen as the experimental model because its function is far better defined than that of any other LGIC, allowing one to interpret anesthetic actions within the framework of a well-established and robust kinetic model. The method used to define anesthetic actions on the nAcChoR is a new rapid sequential mixing stopped-flow fluorescence assay developed and validated by the PI that can assess anesthetic actions on agonist binding, channel gating, and desensitization kinetics without the potentially confounding effects of anesthetic-induced channel blockade.
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