General anesthesia is a condition in which loss of memory and consciousness is accompanied by loss of pain sensation. Inhalation general anesthetics (GA), fundamental aides in surgical anesthesia, induce both but not without displaying some serious side effects. Because of the relevance of neurotransmission in hypnotic and analgesic effects, a substantial effort has been placed in the study of the action of GA at the synapse, with emphasis on effects on ligand-gated receptors and channels. The effects of GA on voltage-gated ion channels are of no less importance in neuronal transmission. Voltage-gated ion channels play an added crucial role in general anesthesia in that many side effects of GA have been traced to impaired function of these channels in various tissues. A well-documented example of GA induced side effects involving voltage-gated channels is that of cardiac depression, which puts patients under considerable risk. The design of 'ideal' general anesthetics will emerge from the knowledge of the specific molecular mechanisms involved in GA effects. It is the long-term goal of this project to contribute to the understanding of the mechanisms of action of GA by providing novel information on G-channel interactions. In this proposal the effects of volatile anesthetics an of a very promising intravenous anesthetic, Propofol, will be studied in detail by taking advantage of the tools provided by molecular biology, by a high expression system (Xenopus oocytes) and by high resolution electrophysiological recording techniques. With this approach, new detailed studies of the function of single species of channels can be performed with essentially no contamination from other membrane proteins.
The specific aims are: 1) To characterize the action of general inhalation anesthetics on the macroscopic gating and conductance properties of cloned voltage-gated ion channels. The cut-open oocyte voltage-clamp technique will be used to record ionic and gating currents from oocytes expressing K+, Na+ or Ca2+ channels from injected cRNA. 2) To characterize general anesthetic effects on the function of the individual channel proteins by recording at the single-channel level. Single-channel activity will be recorded using the patch-clamp technique, 3) To determine, from comparative studies, regions of the structure that interact with the anesthetics and/or that are directly involved in the affected functions. In these studies, different combinations of subunits and channel subtypes., chimeras and channel mutants will be examined. The goal is to identify regions in the proteins involved in specific interactions with the anesthetics. The roles played by auxiliary subunits will be investigated. (4) To initiate studies on the action of intravenous anesthetics on voltage-gated ion channels. These studies will concentrate on the effects of Propofol on macroscopic, gating and single-channel currents. The outcome of this project should provide important information on relevant structural features intervening in GA- channel interactions.
