Abstract
Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand-gated ion channels (pLGICs) and can be effective anesthetics, analgesics, or anti-epileptic drugs. The complex effects of neurosteroids on pLGICs suggest the presence of multiple binding sites in these receptors. Here, using a series of novel neurosteroid-photolabeling reagents combined with top-down and middle-down mass spectrometry, we have determined the stoichiometry, sites, and orientation of binding for 3α,5α-pregnane neurosteroids in the Gloeobacter ligand-gated ion channel (GLIC), a prototypic pLGIC. The neurosteroid-based reagents photolabeled two sites per GLIC subunit, both within the transmembrane domain; one site was an intrasubunit site, and the other was located in the interface between subunits. By using reagents with photoreactive groups positioned throughout the neurosteroid backbone, we precisely map the orientation of neurosteroid binding within each site. Amino acid substitutions introduced at either site altered neurosteroid modulation of GLIC channel activity, demonstrating the functional role of both sites. These results provide a detailed molecular model of multisite neurosteroid modulation of GLIC, which may be applicable to other mammalian pLGICs.
Highlights
Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand-gated ion channels and can be effective anesthetics, analgesics, or anti-epileptic drugs
Neurosteroids photolabel the transmembrane domains (TMDs) of Gloeobacter ligandgated ion channel (GLIC) with a stoichiometry of two To determine the stoichiometry of neurosteroid binding in a pentameric ligand-gated ion channels (pLGICs), we photolabeled purified n-dodecyl--D-maltoside (DDM)-solubilized GLIC with (3␣,5␣)-6-azi-pregnanolone (5␣-6-AziP) (Fig. 1A), a photoreactive analogue of the endogenous neurosteroid allopregnanolone, and analyzed the labeled protein using top-down mass spectrometry (MS)
Neurosteroids are potent endogenous molecules that modulate the activity of neuronal pLGICs and are being developed as anti-epileptics, anti-depressants, general anesthetics, analgesics for chronic pain, and neuroprotective agents [7]
Summary
Neurosteroids photolabel the TMD of GLIC with a stoichiometry of two To determine the stoichiometry of neurosteroid binding in a pLGIC, we photolabeled purified n-dodecyl--D-maltoside (DDM)-solubilized GLIC with (3␣,5␣)-6-azi-pregnanolone (5␣-6-AziP) (Fig. 1A), a photoreactive analogue of the endogenous neurosteroid allopregnanolone, and analyzed the labeled protein using top-down MS. MS analysis of intact photolabeled GLIC demonstrates efficient labeling by 5␣-6-AziP at 100 M (Fig. 1C; Table S1) and at 100 M labeled three times, with a labeling stoichiometry of two (i.e. two neurosteroids per GLIC subunit) (Fig. 1, B and D). Top-down fragmentation by higher-energy collisional dissociation (HCD) of the singly labeled GLIC species yields a series of 5␣-6-AziP-containing y-ions that localize at least one of the labeled sites to the C-terminal end of TM3 or TM4 (Fig. 1E). To search for additional sites, a second allopregnanolone molecule was docked to the GLIC TMD with one allopregnanolone molecule placed within the intersubunit site This yielded additional binding clusters, including one located in an intrasubunit site encompassed by Tyr-194 and Phe-315 (Fig. 3E). Our photolabeling results reveal two putative neurosteroid-binding sites: an intersubunit site between TM3 and TM1 of adjacent subunits and an intrasubunit site between TM1 and TM4
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