title: Manipulations of extracellular Loop 2 in α1 GlyR ultra-sensitive ethanol receptors (USERs) enhance receptor sensitivity to isoflurane, ethanol, and lidocaine, but not propofol.
abstract: We recently developed ultra-sensitive ethanol receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild-type (WT) receptors. The current study investigated: (1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and (2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on α1 GlyRs within the extracellular Loop 2 region.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90089, USA.
authors.forename: A
authors.initials: A
authors.lastname: Naito
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90089, USA.
authors.forename: K H
authors.initials: KH
authors.lastname: Muchhala
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90089, USA.
authors.forename: J
authors.initials: J
authors.lastname: Trang
authors.affiliation: Titus Family Department of Clinical Pharmacy and Pharmaceutical Economics and Policy, University of Southern California, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90089, USA.
authors.forename: L
authors.initials: L
authors.lastname: Asatryan
authors.affiliation: Department of Anesthesia, Beckman Program for Molecular and Genetic Medicine, Stanford University, Stanford University Medical Center, Stanford, CA 94305, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Trudell
authors.affiliation: Department of Anesthesiology, University of Pittsburgh, 6060 Biomedical Science Tower 3, Pittsburgh, PA 15261, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, 6060 Biomedical Science Tower 3, Pittsburgh, PA 15261, USA.
authors.forename: G E
authors.initials: GE
authors.lastname: Homanics
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90089, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Alkana
authors.affiliation: Titus Family Department of Clinical Pharmacy and Pharmaceutical Economics and Policy, University of Southern California, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90089, USA. Electronic address: ddavies@usc.edu.
authors.forename: D L
authors.initials: DL
authors.lastname: Davies
mesh_terms: D000818:Animals; D001703:Biophysics; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D000431:Ethanol / Q000494:pharmacology*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D006993:Hypnotics and Sedatives / Q000494:pharmacology*; D007530:Isoflurane / Q000494:pharmacology; D008012:Lidocaine / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009865:Oocytes; D018408:Patch-Clamp Techniques; D015742:Propofol / Q000494:pharmacology; D018009:Receptors, Glycine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
chemical_list: D002492:Central Nervous System Depressants; D006993:Hypnotics and Sedatives; D018009:Receptors, Glycine; D000431:Ethanol; D008012:Lidocaine; D007530:Isoflurane; D015742:Propofol
keywords: Loop 2; ethanol; isoflurane; lidocaine; ligand-gated ion channel; α1 GlyRs
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