The long term objectives of this project are to identify the mechanisms by which local anesthetics produce their therapeutic and toxic actions. Although several of the primary actions of local anesthetics/antiarrhythmic (ClassI), namely neuronal impulse blockage and anti-fibrillatory actions, are known to be mediated through inhibition of voltage-gated Na channels, these drugs are not very selective and other ion channels and functional membrane proteins are also susceptible to them.
The specific aims of this proposal involve investigations of mechanisms and sites on membrane targets essential for neural transmission, Specific aims: 1. Further detail the binding site, access routes and molecular mechanisms for local anesthetic inhibition of neuronal Na channels: 2. Contrast the pharmacology of local anesthetic direct inhibition of Na channels with that of K+ and Ca2+ channels. 3. Characterize the inhibition by local anesthetics of G-protein coupled neuronal receptors; e.g. Sub P. 4. Use photoaffinity labeling to identify and compare those regions of the target proteins where local anesthetics bind to exert their pharmacological actions. Electrophysiological (patch-clamp) and the biochemical measurements of ionic currents and ligand binding, respectively, will be used to characterized inhibition by LAs. Access and agrees routes to and from ion channels will be investigated by separately changing aqueous viscosity and membrane fluidity. Pharmacological profiles will include dependence of action of drug on a) hydrophobicity and intra-membrane location, b) drug ionization, c) stereoselectivity, d) ion competition and e) state of the target protein. The last aspect considers the ability of voltage, transmitter (agonists), G-proteins and nucleotides, and other drugs to modulate the state (conformation) of the target protein and thereby alter the action of local anesthetics. It is likely that, at concentrations used clinically for local regional anesthesia, local anesthetics affect a variety of membrane proteins involved in signal transmission. These studies will inform us about these actions and provide a more complete description of the clinical situation, thus permitting more thoughtful design of these drugs for different procedures.
