Person: Miller, Keith
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Miller
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Keith
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Miller, Keith
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Publication High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ subunits(Public Library of Science, 2018) Zhou, Xiaojuan; Desai, Rooma; Zhang, Yinghui; Stec, Wojciech; Miller, Keith; Jounaidi, YoussefThe inhibitory γ-aminobutyric acid type A receptors are implicated in numerous physiological processes, including cognition and inhibition of neurotransmission, rendering them important molecular targets for many classes of drugs. Functionally, the entire GABAAR family of receptors can be subdivided into phasic, fast acting synaptic receptors, composed of α-, β- and γ-subunits, and tonic extrasynaptic receptors, many of which contain the δ-subunit in addition to α- and β-subunits. Whereas the subunit arrangement of the former group is agreed upon, that of the αβδ GABAARs remains unresolved by electrophysiological and pharmacological research. To resolve such issues will require biophysical techniques that demand quantities of receptor that have been previously unavailable. Therefore, we have engineered a stable cell line with tetracycline inducible expression of human α4-, β3- and N-terminally Flag-tagged δ-subunits. This cell line achieved a specific activity between 15 and 20 pmol [3H]muscimol sites/mg of membrane protein, making it possible to obtain 1 nmole of purified α4β3δ GABAAR from sixty 15–cm culture dishes. When induced, these cells exhibited agonist–induced currents with characteristics comparable to those previously reported for this receptor and a pharmacology that included strong modulation by etomidate and the δ-subunit-specific ligand, DS2. Immunoaffinity purification and reconstitution in CHAPS/asolectin micelles resulted in the retention of equilibrium allosteric interactions between the separate agonist, anesthetic and DS2 sites. Moreover, all three subunits retained glycosylation. The establishment of this well–characterized cell line will allow molecular level studies of tonic receptors to be undertaken.Publication Sensitivity to Sevoflurane anesthesia is decreased in mice with a congenital deletion of Guanylyl Cyclase-1 alpha(BioMed Central, 2017) Nagasaka, Yasuko; Wepler, Martin; Thoonen, Robrecht; Sips, Patrick Y.; Allen, Kaitlin; Graw, Jan A.; Yao, Vincent; Burns, Sara M.; Muenster, Stefan; Brouckaert, Peter; Miller, Keith; Solt, Ken; Buys, Emmanuel; Ichinose, Fumito; Zapol, WarrenBackground: Volatile anesthetics increase levels of the neurotransmitter nitric oxide (NO) and the secondary messenger molecule cyclic guanosine monophosphate (cGMP) in the brain. NO activates the enzyme guanylyl cyclase (GC) to produce cGMP. We hypothesized that the NO-GC-cGMP pathway contributes to anesthesia-induced unconsciousness. Methods: Sevoflurane-induced loss and return of righting reflex (LORR and RORR, respectively) were studied in wild-type mice (WT) and in mice congenitally deficient in the GC-1α subunit (GC-1−/− mice). Spatial distributions of GC-1α and the GC-2α subunit in the brain were visualized by in situ hybridization. Brain cGMP levels were measured in WT and GC-1−/− mice after inhaling oxygen with or without 1.2% sevoflurane for 20 min. Results: Higher concentrations of sevoflurane were required to induce LORR in GC-1−/− mice than in WT mice (1.5 ± 0.1 vs. 1.1 ± 0.2%, respectively, n = 14 and 14, P < 0.0001). Similarly, RORR occurred at higher concentrations of sevoflurane in GC-1−/− mice than in WT mice (1.0 ± 0.1 vs. 0.8 ± 0.1%, respectively, n = 14 and 14, P < 0.0001). Abundant GC-1α and GC-2α mRNA expression was detected in the cerebral cortex, medial habenula, hippocampus, and cerebellum. Inhaling 1.2% sevoflurane for 20 min increased cGMP levels in the brains of WT mice from 2.6 ± 2.0 to 5.5 ± 3.7 pmol/mg protein (n = 13 and 10, respectively, P = 0.0355) but not in GC-1−/− mice. Conclusion: Congenital deficiency of GC-1α abolished the ability of sevoflurane anesthesia to increase cGMP levels in the whole brain, and increased the concentration of sevoflurane required to induce LORR. Impaired NO-cGMP signaling raises the threshold for producing sevoflurane-induced unconsciousness in mice.Publication The Location and Nature of General Anesthetic Binding Sites on the Active Conformation of Firefly Luciferase; A Time Resolved Photolabeling Study(Public Library of Science, 2012) Shanmugasundararaj, Sivananthaperumal; Lehle, Simon; Yamodo, Herve I.; Husain, S. Shaukat; Tseng, Claire; Nguyen, Khanh; Addona, George H.; Miller, KeithFirefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP–driven production of light. We have used time–resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 \(\mu\)M, close to its general anesthetic potency. Photoincorporation of [\(^3\)H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC–MSMS revealed a similar conformation–dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.Publication Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer(Springer Nature, 2019-01) Laverty, Duncan; Desai, Rooma; Uchański, Tomasz; Masiulis, Simonas; Stec, Wojciech J.; Malinauskas, Tomas; Zivanov, Jasenko; Pardon, Els; Steyaert, Jan; Miller, Keith; Aricescu, A. RaduType A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated ion channels (pLGICs) and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system. Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia. Amongst the numerous assemblies theoretically possible, α1β2/3γ2 GABAARs are most prevalent in the brain. The β3 subunit plays an important role in maintaining inhibitory tone and expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in a CRISPR/Cas9 derived β1-3 triple knockout. To date, efforts to generate accurate structural models for heteromeric GABAARs have been hampered by the use of engineered receptors and the presence of detergents. Significantly, some recent cryo-EM reconstructions report collapsed conformations which disagree with the prototypical pLGIC, the Torpedo nicotinic acetylcholine receptor, the large body of structural work on homologous homopentameric receptor variants, and the logic of an ion channel architecture. To address this problem, here we present a high-resolution cryo-EM structure of the full-length human α1β3γ2L, a major synaptic GABAAR isoform, functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator megabody and in a desensitized conformation. Unexpectedly, each GABAAR pentamer harbours two phosphatidylinositol 4,5-bisphosphate (PIP2) molecules, whose head groups occupy positively-charged pockets in the intracellular juxtamembrane regions of α1-subunits. Beyond this level, the intracellular M3-M4 loops are largely disordered, possibly because interacting post-synaptic proteins were not included. This structure illustrates the molecular principles of heteromeric GABAA receptor organization and provides the reference framework for future mechanistic investigations of GABA-ergic signaling and pharmacology.Publication GABAA receptor signalling mechanisms revealed by structural pharmacology(Springer Nature, 2019-01) Masiulis, Simonas; Desai, Rooma; Uchański, Tomasz; Serna Martin, Itziar; Laverty, Duncan; Karia, Dimple; Malinauskas, Tomas; Zivanov, Jasenko; Pardon, Els; Kotecha, Abhay; Steyaert, Jan; Miller, Keith; Aricescu, A. RaduType-A -aminobutyric receptors (GABAARs) are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and common substances of abuse. Without reliable structural data, the mechanistic basis for pharmacological modulation of GABAARs remains largely unknown. Here we report high-resolution cryoEM structures of the full-length human 132L GABAAR in lipid nanodiscs, bound to the channel blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA and the classical benzodiazepines alprazolam (Xanax) and diazepam (Valium), respectively. We describe the binding modes and mechanistic impacts of these ligands, the closed and desensitised states of the GABAAR gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding, and the transmembrane, pore-forming, regions. This work provides a structural framework to integrate decades of physiology and pharmacology research and a rational basis for development of novel GABAAR modulators.