Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABA(A) receptor

Abstract

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-β3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-β3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-β3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the β3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3β,5β)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-β3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole β3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

Fig. 1.

6-AziP modulation of [³⁵S]TBPS binding to β3 homomeric GABA_A receptors in Sf9 cell membranes, rat brain membranes, and α1β2 GABA_A receptors expressed in TSA cells. 6-AziP produced one-component inhibition of [³⁵S]TBPS binding to the recombinant His₈-β3 homomeric GABA_A receptors (IC₅₀ = 9.0 ± 1 μM), whereas it produced two-component (enhancement and inhibition) modulation with rat brain membranes and α1β2 receptors.

Fig. 2.

6-AziP photolabeling of His₈-β3 homomeric GABA_A receptors. Sf9 cell membranes (2 mg of protein) expressing His₈-β3 homomeric GABA_A receptors were photolabeled with 30 μM [³H]6-AziP, purified with a nickel affinity column, and analyzed through SDS-PAGE. A, the purified proteins were immunoblotted with bd17 antibody, which showed that the β3 subunits were purified as monomers, dimers, and trimers. B, the lysate (lane L), the flow-through fraction from the column (lane F), and purified proteins (lane E) were stained with SYPRO Ruby. C, scintillation counting of gel slices showed the protein bands photolabeled with [³H]6-AziP. Stars, major photolabeled form of the β3 subunit.

Fig. 3.

Stereoselectivity of photoincorporation of [³H]6-AziP into β3 homomeric GABA_A receptors. Five hundred micrograms of Sf9 cell membranes expressing His₈-β3 homomeric GABA_A receptors were photolabeled with 30 μM [³H]6-AziP [(3α,5β)-6-AziP] or its isomer [³H](3β,5β)-6-AziP. A, radioactivity in slices from SDS-PAGE of photolabeled β3 membranes. Inset, the 3α-isomer of 6-AziP labeled a 55-kDa protein, whereas the 3β-isomer did not. B, radioactivity in gel slices from SDS-PAGE of His₈-β3 receptors purified on a nickel affinity column after photolabeling with either the 3α- or 3β-isomer of 6-AziP. The 3α-isomer labeled the 55-kDa protein (β3 subunit) twice as effectively did as the 3β-isomer.

Fig. 4.

Flowchart for identification of the 6-AziP photolabeling site through mass spectrometry. Membranes from Sf9 cells expressing His₈-β3 homomeric GABA_A receptors were photolabeled with 15 μM [³H]6-AziP or irradiated with UV light in the presence of pregnanolone (15 μM). The His₈-β3 homomeric GABA_A receptors were then purified with a nickel affinity column and subjected to timed, in-solution digestion with several endoproteases. Peptides were recovered through sequential solid-phase extractions with C₄ and PGC and were analyzed through high-resolution (HR) nano-LC-MS with electrospray ionization. O/N, overnight.

Fig. 5.

Identification of a 6-AziP-modified peptide in TM3 of the His₈-β3 subunit of the GABA_A receptor. A, MS2 mass spectrum of ALLEYAF-6-AziP, a Lys-C/chymotrypsin-digested peptide from the TM3 segment of the GABA_A receptor β3 subunit with a 6-AziP adduct on Phe301. The MS2 spectrum was acquired from an [M+2H]²⁺ ion (inset). B, XICs of ALLEYAF-6-AziP (m/z 571.851) from timed Lys-C/chymotrypsin digests of photolabeled and nonphotolabeled β3 subunits aligned according to retention times and m/z values by using Rosetta Elucidator. The XIC of ALLEYAF-6-AziP (m/z 571.851 and retention time of 49–51 min) was observed in all of the chymotrypsin digests from photolabeled samples and in none of the digests from nonphotolabeled samples.

Fig. 6.

Mass spectrometric sequence coverage of the β3 subunit of the GABA_A receptor. Five micrograms of Sf9 cell membranes expressing His₈-β3 homomeric GABA_A receptors were photolabeled with 15 μM 6-AziP. The receptors were purified with a nickel affinity column and were analyzed through mass spectrometry after Lys-C/trypsin or Lys-C/chymotrypsin digestion. Ninety percent of the peptide sequence was detected, as shown in red. Frames indicate the TMDs of the protein.

Fig. 7.

Structural modeling of homomeric β3 GABA_A receptors. A and C, β3 GABA_A receptor models using the Glu-Cl X-ray-determined structure (A) and the nicotinic acetylcholine receptor cryo-electron microscopy-determined structure (C) as templates. B and D, enlarged view of the transmembrane regions of two adjacent subunits, showing candidate 6-AziP-interacting amino acids with Glu-Cl (B) and nicotinic acetylcholine receptor (D) as templates. In all panels, Phe301 on one subunit is colored magenta; in B and D, all 6-AziP-accessible residues within 9 Å of Phe301 are shown in stick form and residues located on TM4 are colored orange, TM3 blue, and TM1 and TM4 of the adjacent subunit purple. Inset, structure of 6-AziP with only polar hydrogens shown. The distance from C6 to the ketone oxygen is 8.42 Å; this distance was rounded to 9 Å and was used to identify the candidate 6-AziP-accessible residues shown in B and D.