Volatile anesthetics (Vas) are invaluable aids in surgical anesthesia, yet serious side effects complicate their use. An understanding of the fundamental mechanisms underlying VA action will contribute to the development of more specifically targeted, and thereby safer agents. The clinically essential site of VA action is the central nervous system (CNS). The molecular mechanism of action of VAs in the CNS remains controversial. The effects of such agents in the CNS may be explained in part by their ability to potentiate neuronal inhibition mediated by the neurotransmitters gamma-aminobutyric acid (GABA) and glycine at GABAa and glycine receptors, respectively. Ligand-gated chloride channel receptors exhibit differential sensitivities to the VAs; whereas GABAa and glycine receptors are positively modulated, GABAc receptors are negatively modulated. Such differences suggest that there may be a structural basis for interactions with VAs. Halothane and other halogenated alkane VAs appear to have a different site of action in these receptors, when compared to VA ethers, such as isoflurane and enflurane, or alcohol. This is consistent with data supporting multiple sites of VA action. The goal of this research is to elucidate the molecular site(s) of action of halothane and related VAs in GABAa and glycine receptors using the tools of molecular biology and electrophysiology. The hypotheses are that (a) there are specific sites at which halothane and related VAs interact with these receptors, and (b) these interactions are subunit dependent.
The specific aims are: 1) to generate, and study the pharmacology of, chimeric receptors by interchanging portions of GABAa, glycine and GABAc subunits, to identify specific domains of the protein involved in sensitivity to these VAs. Two electrode voltage clamp (TEVC) will be used to record currents from Xenopus oocytes expressing chimeras. 2) to generate, and study the pharmacology of, point-mutated GABAa,glycine and GABAC subunits, to demonstrate that sensitivity to such VAs requires interaction with specific amino acid residues of these proteins. Mutants will be expressed in oocytes and studies by TEVC. 3) to replicate point mutants of interest in other GABAa and glycine subunits, and to study the pharmacology of various subunit combinations, defining the role of subunit dependence in the effects of halothane and related VAs at these sites. These studies will provide insights into the molecular interaction of this group of Vas with specific neuronal components, which may facilitate the design of improved general anesthetics.
| Greenblatt, E P; Meng, X (2001) Divergence of volatile anesthetic effects in inhibitory neurotransmitter receptors. Anesthesiology 94:1026-33 |
| Jenkins, A; Greenblatt, E P; Faulkner, H J et al. (2001) Evidence for a common binding cavity for three general anesthetics within the GABAA receptor. J Neurosci 21:RC136 |
