Three classes of propofol binding sites on GABAA receptors

Abstract

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator of gamma-aminobutyric acid type A (GABA_A) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. Ortho-propofol diazirine (o-PD) labels β-H267, a pore-lining residue, whereas AziPm labels residues β-M286, β-M227, and α-I239 in the two membrane-facing interfaces [β(+)/α(-) and α(+)/β(-)] between α and β subunits. This study used photoaffinity labeling of α₁β₃ GABA_A receptors to reconcile the apparently conflicting results obtained with AziPm and o-PD labeling, focusing on whether β₃-H267 identifies specific propofol binding site(s). The results show that propofol, but not AziPm protects β₃-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of β₃-M286, β₃-M227, and α₁I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in β₃ homomeric receptors, demonstrated a minimum of five AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per β-subunit. The native mass spectrometry data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified by o-PD are adjacent to, but not within the channel pore, since the pore at the 17' H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific propofol binding sites on α₁β₃ GABA_A receptors.

Keywords: fluorescence resonance energy transfer (FRET); gamma-amino butyric acid (GABA); ligand binding protein; mass spectrometry (MS); neurotransmitter receptor; steroid.

Figure 1

Propofol binding sites predicted by photolabeling on an α₁β₃GABA_Areceptor. A, side view of an α₁β₃ GABA_A receptor (PDB: 7PBD) with transmembrane domain helices illustrated as cylinders. The α₁ subunits are shown in yellow and the β₃ subunit in cyan. Propofol molecules are illustrated as ovals with the orange propofol in the α₁(+)/β₃(−) membrane-adjacent site, the purple propofol in the pore-adjacent site and the salmon propofol in the β₃(+)/α₁(−) membrane-adjacent site. B, magnified side view of binding sites viewed from the membrane face of the receptor with propofol molecules docked in their energetically preferred pose in each binding site. The membrane-adjacent sites are approximately two α-helical turns below the pore-adjacent site. Residues photolabeled by propofol analogues are shown in stick format. C, vertical view of the α₁β₃ GABA_A receptor from the extracellular side illustrating that the putative membrane-adjacent propofol binding sites are on the outside of the receptor while the putative pore-adjacent sites are on the pore-facing side. D, magnified extracellular vertical view with propofol molecules docked in their energetically preferred pose in each site. E–H, molecular structures of propofol and the photolabeling analogues o-PD, AziPm, and AziPm-d₉. GABA_A, gamma-aminobutyric acid type A; o-PD, ortho-propofol diazirine; PDB, Protein Data Bank.

Figure 2

o-PD photolabeling of the H267 residue on β₃-TM2 of an α₁β₃GABA_Areceptor is inhibited by propofol, but not AziPm-d. A, fragment ion spectrum of o-PD (10 μM) photolabeling of H267 on the β₃-TM2 peptide ²⁵¹VALGITTVLTMTTINTHLRETLPK²⁷⁴. The y8∗-y13∗ fragment ions (red∗) contain the o-PD adduct, whereas the y6 and y7 fragment ions (black) do not, indicating H267 as the photolabeled residue. The unlabeled (black) y8 and y9 ions represent neutral loss. B, photolabeling of the β₃-TM2 peptide by o-PD (10 μM) in the presence and absence of propofol (300 μM) or AziPm-d (300 μM). Propofol but not AziPm-d prevented o-PD photolabeling. Results were compared using a two-tailed paired t test. ∗∗ = p < 0.01. GABA_A, gamma-aminobutyric acid type A; o-PD, ortho-propofol diazirine; TM, transmembrane.

Figure 3

AziPm photolabeling of membrane-adjacent sites in α₁β₃GABA_Areceptors is inhibited by both propofol and o-PD. A, fragment ion spectrum of β₃-TM3 peptide photolabeled by AziPm on M286 as identified by site defining ions b6 and b7∗ (fragment ions containing the adduct are shown in red with a star; ions not containing the adduct are shown in black). B, fragment ion spectrum of α₁-TM1 photolabeled by AziPm on I239 as identified by site defining ions y10 and y11∗. C, overlay of the fragment ion spectra of β₃-TM1 peptide photolabeled by AziPm and AziPm-d₇ on M227 as identified by site defining ions b10 and b11∗. The inset illustrates that fragment ions not containing the adduct (b10/b10) are identical in the AziPm and AziPm-d₇ spectra, whereas fragment ions containing the adduct (b11^AziPm/b11^AziPm-d7) differ by 7 da. Precursor ions with an AziPm-d₈ and AziPm-d₉ adduct were also detected; in fragmentation spectra of these precursors, fragment ions containing the adduct differed by 8 or 9 mass units from the AziPm adduct, respectively. D, photolabeling of β₃-TM3, α₁-TM1, and β₃-TM1 peptides by AziPm is reduced in the presence of propofol (1 mM). E, photolabeling of β₃-TM3, α₁-TM1, and β₃-TM1 peptides by AziPm-d is reduced in the presence of o-PD (300 μM). Data in panels D and E were analyzed using a paired t test. ∗ = p < 0.05. GABA_A, gamma-aminobutyric acid type A; o-PD, ortho-propofol diazirine; TM, transmembrane.

Figure 4

Computational docking of propofol, o-PD, and AziPM in the pore-adjacent and membrane-adjacent sites in an α₁β₃GABA_Acryogenic electron microscopy structure (PDB:7PBD). A, top-down view (from an extracellular perspective) showing the most energetically favorable poses of propofol (pink) and o-PD (blue) docked in the pore-adjacent site. The α₁ subunit is shown in beige and the β₃ subunit in cyan. Residues β₃-Y143, -F221, and -Q224 in the binding pocket are shown in yellow stick and ball format; the H267 residue photolabeled by o-PD is shown in purple. AziPm is not shown because it did not dock in this site. B, side view (from membrane perspective) of propofol and o-PD docked in the pore-adjacent site. C and D, side views (from the membrane perspective) of propofol (pink), o-PD (blue), and AziPm (orange) docked in their most energetically favorable poses in the: (C) β₃(+)/α₁(−) and; (D) α₁(+)/β₃(−) membrane-adjacent sites. Photolabeled residues are shown in stick and ball format. GABA_A, gamma-aminobutyric acid type A; o-PD, ortho-propofol diazirine; PDB, Protein Data Bank.

Figure 5

Native mass spectrometric analysis of photolabeling stoichiometry in homomeric β₃GABA_Areceptors. A, AziPm labeling: (Top panel) spectrum of unlabeled β₃-ILC-del-homopentamer showing three charge-states (+27, +28, and +29). The calculated molecular mass of the pentamer is 215 kDa. (Lower panel) spectrum of β₃-homopentamer photolabeled three times with 200 μM AziPm showing charge states +27, +28, and +29. Charge-state +28 shows 6 features, corresponding to the unlabeled receptor (dashed line) and five more features each with an additional mass of 216 ± 17 Da, consistent with AziPm adducts. B, same spectra as (A) focused on charge-state +28. The dashed line indicates the unlabeled β₃-homopentamer and the numbers in red indicate the number of AziPm adducts in a β₃-homopentamer.C, o-PD labeling: (Top Panel) spectrum of unlabeled β₃-ICL-del homopentamer showing three charge-states (+29, +30, and +31) each corresponding to a molecular mass of 215 kDa. (Lower panel) spectrum of β₃-ICL-del homopentamer photolabeled three times with 100 μM o-PD showing charge-states +29, +30, and + 31. Charge-state +30 shows four features, corresponding to the unlabeled receptor (dashed line) and three more features each with an additional mass of 232 ± 42 Da, consistent with o-PD adducts. D, same spectra as (C) focused on charge-state +30. The dashed line indicates the unlabeled β₃-homopentamer and the numbers in red indicate the number of o-PD adducts in a β₃-homopentamer. GABA_A, gamma-aminobutyric acid type A; o-PD, ortho-propofol diazirine.

Figure 6

The effect of ZnCl₂on o-PD photolabeling of β₃-TM2 peptide in α₁β₃β₃-BRILhomomeric GABA_Areceptors. A, ZnCl₂ completely inhibits o-PD photolabeling of β₃-TM2 peptide in α₁β₃ GABA_A receptors. B, ZnCl₂ partially (∼70%) inhibits o-PD photolabeling of β₃-TM2 peptide in β₃-homomeric GABA_A receptors. α₁β₃ GABA_A receptors were labeled with 10 μM o-PD and β₃-homomeric receptors with 30 μM to achieve similar photolabeling efficiency. Samples with and without ZnCl₂ (n = 3 for each) were compared using a unpaired t test with ∗ = p < 0.05. GABA_A, gamma-aminobutyric acid type A; o-PD, ortho-propofol diazirine; TM, transmembrane.

Figure 7

Synthetic scheme for synthesis of cumene-d₁₁from benzene-d₆.