For a century the challenge of the Meyer-Overton Rule has remained. The lipid bilayer provides the best description of the pharmacology of anesthesia, yet no mechanism has emerged from this line of research. Currently, attention is focused on excitable membrane proteins, but it is difficult to prove beyond doubt that anesthetic sites exist on these because of the high degree of nonspecific binding. On the nicotinic receptor indirect evidence for an inhibitory site has been deduced from careful kinetic and site-directed mutagenesis experiments. However, the latter information is difficult to interpret because of allosteric effects. Neither X-ray crystallography nor NMR spectroscopy can be used to locate anesthetic sites on membrane proteins, but photoaffinity labeling offers the chance to locate such sites at the primary structure level. Therefore, we have synthesized and characterized a novel stable general anesthetic, 3-(2-hydroxyethyl)-3-n-pentyldiazirine, or 3-azioctanol that can be activated by light. It anesthetized tadpoles with an EC50 of 160 µM. Sub-anesthetic concentrations of 3-azioctanol enhanced GABA-induced currents in GABAA receptors, an effect that has been implicated in general anesthetic action. In muscle nicotinic acetylcholine receptors (nAcChoR), it inhibited the response to acetylcholine with an IC50 of 33 µM. Actions on both receptors were comparable to octanol.
We also synthesized 3-(2-hydroxyethyl)-3-[4,5-3H2]-n-pentyldiazirine, or [3H]3-azioctanol. This photoincorporated into Torpedo nAcChoR-rich membranes mainly in the a-subunit and the lipid. Pre-incubation with agonist enhanced the photoincorporation into the alpha subunit. Proteolytic digestion of the a-subunit revealed that agonist-independent incorporation occurs in a proteolytic fragment containing the M4 transmembrane segment, which forms part of the lipid-protein interface. Agonist preincubation enhanced the photolabeling 10-fold in the proteolytic fragment containing the M1, M2 and M3 transmembrane segments. Within this fragment the agonist effect is localized on the channel-lining alpha helix (M2). To determine which of these sites is responsible for channel inhibition two strategies are being followed. (1) We are comparing 3-azioctanol's rate of photoincorporation at each site to its rate of channel inhibition using a freeze-clamping method we have developed which is capable of following the kinetics of photolabeling on the millisecond
time scale. (2) The effect of site directed mutagenesis on 3-azioctanol's inhibitory potency both at the amino acid residues photolabeled and at other putative sites which might act allosterically are being determined.
By combining these two approaches a much deeper understanding of the molecular mechanisms of action of general anesthetics will be obtained.
Supported by a grant from the NIGMS, USA (GM 58448).