Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors

Abstract

Most general anesthetics enhance GABA type A (GABA) receptor activity at clinically relevant concentrations. Sites of action of volatile anesthetics on the GABA receptor remain unknown, whereas sites of action of many intravenous anesthetics have been identified in GABA receptors by using photolabeling. Here, we used photoactivatable analogs of isoflurane (AziISO) and sevoflurane (AziSEVO) to locate their sites on αβγ and αβ GABA receptors. As with isoflurane and sevoflurane, AziISO and AziSEVO enhanced the currents elicited by GABA. AziISO and AziSEVO each labeled 10 residues in αβ receptors and 9 and 8 residues, respectively, in αβγ receptors. Photolabeled residues were concentrated in transmembrane domains and located in either subunit interfaces or in the interface between the extracellular domain and the transmembrane domain. The majority of these transmembrane residues were protected from photolabeling with the addition of excess parent anesthetic, which indicated specificity. Binding sites were primarily located within α+/β- and β+/α- subunit interfaces, but residues in the α+/γ- interface were also identified, which provided a basis for differential receptor subtype sensitivity. Isoflurane and sevoflurane did not always share binding sites, which suggests an unexpected degree of selectivity.-Woll, K. A., Zhou, X., Bhanu, N. V., Garcia, B. A., Covarrubias, M., Miller, K. W., Eckenhoff, R. G. Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors.

Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

“…Sevoflurane and the photolabel used in Ref. (30) both potentiate GABA(A) receptors, which is not consistent with pore block, but sevoflurane is a small, mobile molecule that may occupy the pore transiently without blocking it. We discuss the particular case of pore-binding in a subsequent section.…”

“…Various residues in the intersubunit sites of GABA A R have been identified using photoaffinity labeling as possible binding sites for propofol (29) and sevoflurane (30), as shown in Figure 1. For comparison, the contact frequency and mean distance measured from simulations are listed in Table 1.…”

“…Residues labeled by azisevofluorane (30) are concentrated in the intracellular half of the TMD ( Figure 1D). They are primarily pore-lining residues on the M2 helix, with some on M1 facing the lipid membrane or the intrasubunit cavity.…”

“…They are primarily pore-lining residues on the M2 helix, with some on M1 facing the lipid membrane or the intrasubunit cavity. Since there are no residues labeled on M3, it is not possible to distinguish intersubunit vs intrasubunit binding modes, on the basis of residues labeled in Reference (30) alone. Interpretation of the photolabeling results in (30) relied heavily on complementary docking calculations based on the present structural model of the ↵ 1 3 2 GABA(A) receptor.…”

“…The center of the intersubunit or pore binding cavities for propofol indicated by refinement simulations are marked with gray and cyan spheres, respectively. C-D) Sevoflurane binding site residues indicated through photolabeling with Azisevoflurane (30) are shown in orange. The center of the intersubunit or pore binding cavities for sevoflurane indicated by refinement simulations are marked with gray and cyan spheres, respectively.…”