Although extensive investigations suggest that clinically relevant actions of anesthetics might arise primarily from interactions of anesthesia molecules with excitable proteins in the central nervous system (CNS), the characteristics and the molecular nature of such interactions remain largely unknown. In this proposal, interactions of anesthesia molecules with proteins will be studied in system where two well-characterized, structurally quantifiable channel forming peptides serve as simplified models of transmembrane proteins. We intend to first extend our current studies with gramicidin A, a 15-amino-acid cation channel peptide with highly resolved three-dimensional functional structure. The knowledge gained from the gramicidin A studies will then be used to carry out studies of M2-delta, a peptide that is structurally slightly more complex, but also clinically more relevant. M2-delta is a 23-amino-acid homologous mix of the channel-lining transmembrane segment (the M2 domain) of a superfamily of ligand-gated ion channels known to be important in the action of general anesthetics. The emphasis of the research will be on characterizing basic principles that can be extrapolated to studies of other functional channel proteins. State-of-the-art nuclear magnetic resonance (NMR) spectroscopy will be used to focus on three specific aims that will provide useful, new information not readily available from other models or methods.
Aim 1 : to identify basic, unifying characteristics of specific anesthetic interactions with channel-forming peptides.
Aim 2 : to determine dose- dependent modulations of anesthetics (but not non-anesthetics) on peptide- solvent interactions, and on the secondary structure of the channels.
Aim 3 : to relate functional activity of model channel proteins to direct anesthetic-peptide interactions identified in Specific Aim 1, and to anesthetic-induced conformational changes found in Specific Aim 2. Specifically, the site-selective truncated driven nuclear Overhauser effect (TNOE) will be used to quantify site-selective interactions between anesthetics and peptide residues. High-resolution, two-dimensional nuclear Overhauser effect spectroscopy (NOESY) will be used to analyze structural consequences of anesthetic interactions. Magnetization inversion transfer (MIT) experiments will be conducted to investigate the functional consequences of anesthetic-peptide interaction. Three types of fluorinated anesthetics (i.e., linear and cyclic fluoroalkanes, and fluoroethers) will be used at clinically relevant concentrations; and their non-anesthetic analogues will also be used to test if results found with anesthetics apply only to molecules having anesthetic action.
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