GABAA Receptors and Etomidate
(supported by NIH grant P01GM58448)
Etomidate is a general anesthetic drug that has attractive characteristics for exploring its mechanism in detail. One such feature is stereoselectivity (see Figure 1). R(+)-etomidate is about 20-fold more potent than S(-)-etomidate in animals. Etomidate is one of the most potent general anesthetics in use, producing effects at 2-3 µM (micromolar) concentrations. It turns out that etomidate also has a very limited set of physiological targets. GABAA receptors that contain b2 and b3 subunits are positively modulated by etomidate, but other ligand-gated ion channels are unaffected at clinically relevant concentrations. High concentrations of etomidate can also directly activate GABAA receptors--that is, etomidate is a GABAA receptor agonist. However, etomidate doesn't act via the GABA (orthosteric) site--it is an allosteric agonist. Both the positive modulation of GABA-mediated receptor activation and direct receptor activation by etomidate display stereoselectivity similar to that observed in animals. This observation suggests that etomidate acts via a protein site (rather than lipids) and that it produces general anesthesia largely via its modulation of GABAA receptors. This hypothesis was confirmed by transgenic animal experiments. A single amino-acid mutation at position 265 in the b2 or b3 subunits of GABAA receptors can eliminate etomidate sensitivity in vitro. Rachel Jurd and colleagues created a knock-in mouse containing one of these mutations (N265M) in the GABAA b3 subunit, and this mouse was insensitive to etomidate at 2-3 times the concentration that anesthetized wild-type mice.
Another hypothesis that emerged from the similar stereoselectivity of GABA enhancement and direct receptor activation was that both of these actions might be due to etomidate interactions with identical sites on GABAA receptors. We tested this idea by carefully measuring both actions electrophysiologically in a1b2g2L GABAA receptors expressed in both Xenopus oocytes and HEK293 cells (Rüsch et al, 2004). Based on GABA modulation measured at a single GABA concentration, others had concluded that the different etomidate concentrations that produced GABA modulation versus direct activation implied different sites. In our studies we used GABA response curve leftward shifts as a measure of GABA modulation, and found that there was no evidence for a high-affinity modulation site?in fact the etomidate concentrations that maximally modulate GABAA receptors are indistinguishable from those that produce maximal direct receptor activation. A simple equilibrium model that explains this behavior is a Monod-Wyman-Changeux Allosteric Co-Agonist Model. This model quantitatively explains both direct activation and enhanced GABA responses in the presence of etomidate, based on a single class of etomidate sites that are linked to the receptor's open-closed (gating) equilibrium. Our analysis also suggests that there are two etomidate sites per receptor.
The Allosteric Co-agonist Model, which is determined by only 5 equilibrium parameters, represents a powerful tool for analyzing the impact of receptor or ligand structural changes. For example, we have examined the impact of replacing b2 subunits with b1 subunits. Preliminary data analysis shows that GABA binds with about equal affinity and gates with about equal efficacy in a1b2g2L and a1b1g2L receptors. Etomidate also binds with similar affinity to the resting states of these receptors, but its affinity for the open-channel state (its efficacy in the MWC model) is 50 times higher when b2 subunits are present. A critical implication of this result is that understanding how etomidate works will require understanding its interactions with the open-channel state. Other sites where etomidate is hypothesized to interact with GABAA receptors will also be analyzed using mutagenesis, electrophysiology, and our allosteric co-agonist model.
In collaboration with Professor Keith Miller at Mass General, Richard Olsen at UCLA, and Jon Cohen at Harvard Medical School, experiments are proceeding to identify the amino acids that etomidate contacts in GABAA receptors isolated from cow brains. These use a photolabel derivative of etomidate, azi-etomidate.
We have recently shown that azi-etomidate can irreversibly alter the function of GABAA receptors when bound to active receptors and exposed to UV light. The functional effects of covalent binding with azi-etomidate are similar to the irreversible effects of etomidate binding. Thus, the etomidate site is photo-labeled by azi-etomidate, and the biochemical data from photolabeling experiments will likely identify the etomidate site.
Kinetic studies have demonstrated that etomidate dramatically slows deactivation of GABAA receptor currents that are stimulated with GABA. We have extended the MWC allosteric modeling concepts to create a kinetic model that closely simulates GABA activation, desensitization, and deactivation. We plan to extend that model to incorporate etomidate modulation as well.
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