Protein crystallography under xenon and nitrous oxide pressure: comparison with in vivo pharmacology studies and implications for the mechanism of inhaled anesthetic action

Abstract

In contrast with most inhalational anesthetics, the anesthetic gases xenon (Xe) and nitrous oxide (N(2)O) act by blocking the N-methyl-d-aspartate (NMDA) receptor. Using x-ray crystallography, we examined the binding characteristics of these two gases on two soluble proteins as structural models: urate oxidase, which is a prototype of a variety of intracellular globular proteins, and annexin V, which has structural and functional characteristics that allow it to be considered as a prototype for the NMDA receptor. The structure of these proteins complexed with Xe and N(2)O were determined. One N(2)O molecule or one Xe atom binds to the same main site in both proteins. A second subsite is observed for N(2)O in each case. The gas-binding sites are always hydrophobic flexible cavities buried within the monomer. Comparison of the effects of Xe and N(2)O on urate oxidase and annexin V reveals an interesting relationship with the in vivo pharmacological effects of these gases, the ratio of the gas-binding sites' volume expansion and the ratio of the narcotic potency being similar. Given these data, we propose that alterations of cytosolic globular protein functions by general anesthetics would be responsible for the early stages of anesthesia such as amnesia and hypnosis and that additional alterations of ion-channel membrane receptor functions are required for deeper effects that progress to "surgical" anesthesia.

FIGURE 1

Omit maps of the two gases within the two proteins. Omit maps showing the position of the Xe atom (A) or of the two N₂O molecules (B) within urate oxidase binding site cavity. Omit maps showing the position of the Xe atom (C), the first N₂O molecule (D), and the second N₂O molecule (E) in annexin V. (Contour levels at 3 SD above the average background.)

FIGURE 2

Urate oxidase binding sites for Xe or N₂O. Smooth carbon α-chain representation of urate oxidase monomer with the hydrophobic gas-binding site at close proximity to the active site pocket occupied by the competitive inhibitor 8-azaxanthin. One atom of Xe or two N₂O molecules bind within the hydrophobic cavity. Oxygen, nitrogen, and Xe atoms are colored in red, blue, and orange, respectively.

FIGURE 3

Annexin V hinge movement and binding sites of Xe or N₂O. Smooth carbon α-chain representation of annexin V monomer in low- and high-calcium conformation (colored in purple and blue, respectively) showing the hinge movement of the loop carrying the tryptophan 185 in parallel to the glutamate 226 switch. Both Xe and N₂O bind within the hydrophobic cavity left vacant by the movement of the tryptophan. A second molecule of N₂O binds in the center of the first domain. The color code is the same as above plus the calcium colored in pink. Figs. 2 and 3 were produced with the visualization software InsightII (Accelrys, San Diego, CA).