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

Abstract

Most general anesthetics enhance GABA type A (GABA_A) receptor activity at clinically relevant concentrations. Sites of action of volatile anesthetics on the GABA_A receptor remain unknown, whereas sites of action of many intravenous anesthetics have been identified in GABA_A receptors by using photolabeling. Here, we used photoactivatable analogs of isoflurane (AziISO) and sevoflurane (AziSEVO) to locate their sites on α₁β₃γ_2L and α₁β₃ GABA_A receptors. As with isoflurane and sevoflurane, AziISO and AziSEVO enhanced the currents elicited by GABA. AziISO and AziSEVO each labeled 10 residues in α₁β₃ receptors and 9 and 8 residues, respectively, in α₁β₃γ_2L 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.

Keywords: crosslinking; isoflurane; photoaffinity labeling; sevoflurane.

Figure 1

ISO, AziISO, SEVO, and AziSEVO α₁β₃γ_2L GABA_A receptor activity. A) Membrane view of the α₁β₃γ₂ GABA_A receptor homology model and schematic of the cross-section as indicated by the gray dashed line of the receptor transmembrane domain viewed from the synaptic cleft. Receptor is colored by subunit type (α₁, salmon; β₃, yellow; and γ₂, slate), and membrane-spanning helices (M1–M4) are labeled accordingly for each subunit. The plus and minus subunit interfaces are indicated for each subunit, and the pore (P) is indicated as a gray circle. B) Chemical structures of volatile anesthetics and their photolabel analogs. C) Representative traces of evoked current by GABA EC₁₀ control and after combined GABA EC₁₀ and 0.3 mM ISO, AziISO, SEVO, and AziSEVO exposures within individual X. laevis oocytes expressing the α₁β₃γ_2L GABA_A receptor. D) Fold potentiation of GABA EC₁₀ by 0.3 mM ISO, AziISO, SEVO, and AziSEVO within X. laevis oocytes expressing the α₁β₃γ_2L GABA_A receptor. Data were analyzed by Mann–Whitney U test that compared the evoked currents of the parent anesthetic with the corresponding photolabel analog. No significant differences were observed.

Figure 2

Coverage map for FLAG-α₁β₃GABA_A receptor mass spectrometry analysis for photolabeled samples with ISO (A) and SEVO (B). Sequence of the purified FLAG-α₁β₃ GABA_A receptor with high-confidence coverage in the mass spectrometry analysis denoted as bold residue codes. Transmembrane domain helices are underlined.

Figure 3

Coverage map for FLAG-α₁β₃γ_2L-L3-1D4 GABA_A receptor mass spectrometry analysis for AziISO photolabeled samples without (A) and with (B) ISO. Sequence of the purified FLAG-α₁β₃γ_2L-L3-1D4 GABA_A receptor with high-confidence coverage in the mass spectrometry analysis denoted as bold residue codes. Transmembrane domain helices are underlined.

Figure 4

Coverage map for FLAG-α₁β₃γ_2L-L3-1D4 GABA_A receptor mass spectrometry analysis for AziSEVO photolabeled samples without (A) and with (B) SEVO. Sequence of the purified FLAG-α₁β₃γ_2L-L3-1D4 GABA_A receptor with high-confidence coverage in the mass spectrometry analysis denoted as bold residue codes. Transmembrane domain helices are underlined.

Figure 5

AziISO photolabeled residue within the γ_2L-subunit M1 helix within the α₁β₃γ_2L GABA_A receptor. Mass spectrum of γ_2L-M1′ Y241 AziISO photolabeled peptide. Focused view of the spectrum within ∼700–900 m/z range, with b (red), y (blue), and precursor ions (green) shown (inset). Above the spectrum is the subunit peptide sequence that contains the γ_2L-subunit M1 transmembrane helix. The predicted photolabeled residue is shown in bold, underline, and is indicated by a green asterisk. See the Supplemental Material for associated peptide fragment table.

Figure 6

AziISO photolabeled residues within the α₁-subunits of α₁β₃ and α₁β₃γ_2L GABA_A receptors. Mass spectra of α₁-subunit AziISO photolabeled residues α₁-M2′ I271 (A) and α₁-M2′ P278 (B) within α₁β₃ and α₁β₃γ_2L GABA_A receptors, respectively. Above the spectra are the subunit peptide sequences that contain the α₁-subunit transmembrane helices. Focused view of the spectrum within ∼525–825 (A) and 550–800 m/z (B), with a and/or b (red), y and/or z (blue), and precursor ions (green) shown (inset). Predicted photolabeled residue is shown in bold, underline, and is indicated by a green asterisk. See the Supplemental Material for associated peptide fragment tables.

Figure 7

AziISO photolabeled residues within the β₃-subunits of α₁β₃ and α₁β₃γ_2L GABA_A receptors. Mass spectra of β₃-subunit AziISO photolabeled residues β₃-M3′ V290 (A) and β₃-M2′ I255/β₃-M2′ I264 (B) within α₁β₃ and α₁β₃γ_2L GABA_A receptors, respectively. Focused view the spectrum within ∼350–825 (A) and 280–590 m/z (B), with b (red), y (blue), and precursor ions (green) shown. Predicted photolabeled residues is shown in bold, underlined, and is indicated by a green asterisk. Above the spectra are the subunit peptide sequences that contain the β₃-subunit transmembrane helices. Predicted photolabeled residue is shown in bold, underlined, and is indicated by a green asterisk. See the Supplemental Material for associated peptide fragment tables.

Figure 8

AziSEVO photolabeled residues within the α₁-subunits of α₁β₃ and α₁β₃γ_2L GABA_A receptors. Mass spectra of α₁-subunit AziSEVO photolabeled residues α₁-M1′ C234 (A) and α₁-M1′ S241 (B) within α₁β₃ and α₁β₃γ_2L GABA_A receptors, respectively. Above the spectra are the subunit peptide sequences that contain the α₁-subunit transmembrane helices. Focused view of the spectrum within ∼425–900 (A) and 325–800 m/z (B), with b (red), y (blue), and precursor ions (green) shown. Predicted photolabeled residue is shown in bold, underlined, and is indicated by a magenta asterisk. See the Supplemental Material for associated peptide fragment tables.

Figure 9

AziSEVO photolabeled residues within the β₃-subunits of α₁β₃ and α₁β₃γ_2L GABA_A receptors. Mass spectra of β₃-subunit AziSEVO photolabeled residues β₃-M2′ A249 (A) and β₃-M2′ A248 (B) within α₁β₃ and α₁β₃γ_2L GABA_A receptors, respectively. Above the spectra are the subunit peptide sequences that contain the β₃-subunit transmembrane helices. Focused view of the spectrum within ∼250–600 (A) and 250–600 m/z (B), with b (red), y (blue), and precursor ions (green) shown. Predicted photolabeled residue is shown in bold, underlined, and is indicated by a magenta asterisk. See the Supplemental Material for associated peptide fragment tables.

Figure 10

AziSEVO photolabeled residue within the γ_2L-subunit M1 helix within α₁β₃γ_2L GABA_A receptor. Mass spectra of γ_2L-M2′L268 (A) and γ_2L-M2′G269 (B) AziSEVO photolabeled peptides. Above the spectra is the subunit peptide sequence that contains the γ_2L-subunit M1 transmembrane helix. Focused view of the spectrum within ∼600–1150 (A) and 690–975 m/z (B), with b (red), y (blue), and precursor ions (green) shown. Predicted photolabeled residue is shown in bold, underlined, and is indicated by a magenta asterisk. See the Supplemental Material for associated peptide fragment table.

Figure 11

Docking experiments within predicted ISO binding cavities as indicated by AziISO photolabeled residues in the α₁β₃γ_2L GABA_A receptor. The 3 suggested transmembrane domain binding cavities within the α+/β− (A) and β+/α− (B) interfaces and the α+/γ− intrasubunit/intersubunit cavity (C). All residues that face cavities were made flexible, whereas the backbone structure remained rigid during docking experiments. The gray Connolly surface/mesh dotted representations are of 5 ISO and 5 AziISO in the highest scored poses predicted by AutoDockVina (33). Green spheres indicate the Cα atoms of residues photolabeled by AziISO in the α₁β₃γ_2L GABA_A receptors and are labeled accordingly. The left panel includes a transmembrane domain view of the docking experiments for each unique interface from the synaptic cleft [only 1 of 2 β+/α− interfaces (B) shown]. The bottom panel includes the aligned α₁-, β₃-, and γ_2L-subunit sequences that span the M1–M3 helices, with photolabeled residues colored in green.

Figure 12

Docking experiments within predicted ISO binding cavities as indicated by AziSEVO photolabeled residues in the α₁β₃γ_2L GABA_A receptor. The 4 suggested transmembrane domain binding cavities within the α+/β− (A), β+/α− (B), γ+/β− (C), and α+/γ− (D) interfaces. All residues that face cavities were made flexible, whereas the backbone structure remained rigid during docking experiments. The gray Connolly surface/mesh dotted representations are of 5 ISO and 5 AziSEVO in the highest scored poses predicted by AutoDockVina (33). Magenta spheres indicate Cα atoms of residues photolabeled by AziSEVO in the α₁β₃γ_2L GABA_A receptors and are labeled accordingly. The left panel includes a transmembrane domain view of the docking experiments for each unique interface from the synaptic cleft [only 1 of 2 β+/α− interfaces (B) shown]. The bottom panel includes the aligned α₁-, β₃-, and γ_2L-subunit sequences that span the M1–M3 helices, with photolabeled residues colored in magenta.