PERIOPERATIVE MEDICINE A Single Phenylalanine Residue in the Main Intracellular Loop of ␣1 ␥-Aminobutyric Acid Type A and Glycine Receptors Influences Their Sensitivity to Propofol Gustavo Moraga-Cid, Ph.D.,* Gonzalo E. Yevenes, Ph.D.,* Gu¨ nther Schmalzing, M.D.,† Robert W. Peoples, Ph.D.,‡ Luis G. Aguayo, Ph.D.§ Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/115/3/464/254366/0000542-201109000-00010.pdf by guest on 02 October 2021 ABSTRACT What We Already Know about This Topic • Propofol positively modulates receptors for the inhibitory Background: The intravenous anesthetic propofol acts as a transmitters ␥-aminobutyric acid type A (GABA) and glycine, positive allosteric modulator of glycine (GlyRs) and ␥-ami- but the molecular mechanisms involved are unclear nobutyric acid type A (GABAARs) receptors. Although the role of transmembrane residues is recognized, little is known about the involvement of other regions in the modulatory What This Article Tells Us That Is New effects of propofol. Therefore, the influence of the large in- • A single homologous residue in the large M3-M4 intracellular tracellular loop in propofol sensitivity of both receptors was ␣ loops of the 1 subunits of GABAA and glycine receptors mod- explored. ulates the action of propofol but not of other general anesthetics ␣ ␣ ␤ Methods: The large intracellular loop of 1 GlyRs and 1 2 GABAARs was screened using alanine replacement. Sensitiv- ity to propofol was studied using patch-clamp recording in 80 Ϯ 23%). Remarkably, propofol-hyposensitive mutant re- HEK293 cells transiently transfected with wild type or mu- ceptors retained their sensitivity to other allosteric modula- tant receptors. tors such as alcohols, etomidate, trichloroethanol, and isoflu- Results: Alanine mutation of a conserved phenylalanine rane. At the single-channel level, the ability of propofol to ␣ residue within the 1 large intracellular loop significantly increase open probability was significantly reduced in both Ϯ ␣ Ϯ Ϯ ␣ ␤ reduced propofol enhancement in both GlyRs (360 30 vs. 1 GlyR (189 36 vs. 22 13%) and 1 2 GABAAR Ϯ Ϯ Ϯ Ϯ Ϯ 75 10%, mean SEM) and GABAARs (361 49% vs. (279 29 vs. 29 11%) mutant receptors. Conclusion: In this study, it is demonstrated that the large intracellular loop of both GlyR and GABA R has a con- * Research Assistant, Department of Physiology, University of A Concepcio´n, Concepcio´n, Chile. † Professor, Department of Molec- served single phenylalanine residue (F380 and F385, respec- ular Pharmacology, Rheinisch-Westfaelische Technische Hoch- tively) that influences its sensitivity to propofol. Results sug- schule, Aachen, Germany. ‡ Associate Professor, Department of Biomedical Sciences, Marquette University, Milwaukee, Wisconsin. gest a new role of the large intracellular loop in the allosteric § Professor, Department of Physiology, University of Concepción. modulation of two members of the Cys-loop superfamily. Received from the Department of Physiology, Faculty of Biolog- Thus, these data provide new insights into the molecular ical Sciences, University of Concepcio´n, Concepcio´n, Chile. Submit- framework behind the modulation of inhibitory ion chan- ted for publication April 6, 2010. Accepted for publication May 11, 2011. Supported by grants AA151-50 (L.G.A.) from the National nels by propofol. Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, and FONDECYT 1060368 Chilean Council of Science and Technol- ␥ ogy, Santiago, Chile (L.G.A.). Presented at the Annual Meeting of the LYCINE and -aminobutyric acid receptors (GlyRs Society for Neuroscience, Washington, D.C., November 12–19, G and GABAARs) mediate fast synaptic inhibition in the 2008. central nervous system.1,2 These receptors are members of Address correspondence to Dr. Aguayo: Department of Physi- the Cys-loop receptor family, which share considerable struc- ology, University of Concepcio´n, Barrio Universitario s/n, P.O. Box 3,4 160-C, Concepcio´n, Chile. [email protected]. Information on pur- tural and functional features. As pentamers, they assemble chasing reprints may be found at www.anesthesiology.org or on the around a central pore that transiently opens, allowing the masthead page at the beginning of this issue. ANESTHESIOLOGY’S passive diffusion of anions.3,4 Similar to other members of articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue. the family, their topology consists of an extracellular N-ter- Copyright © 2011, the American Society of Anesthesiologists, Inc. Lippincott minal domain, containing the binding site for the agonist, Williams & Wilkins. Anesthesiology 2011; 115:464–73 four transmembrane domains (TM1-TM4) of which TM2 is Anesthesiology, V 115 • No 3 464 September 2011 PERIOPERATIVE MEDICINE critical for pore formation, and a large intracellular loop con- gating and increased agonist potency, complicating the in- necting TM3 and TM4. To date, molecular cloning has terpretation regarding the reduced propofol sensitivity.13,28 ␣ ␤ identified 5 subunits of the GlyR ( 1–4 and ) and 19 All these studies are in agreement with the idea that resi- ␣ ␤ subunits of the GABAAR, with the GlyR 1 and GABAAR dues in the TM domains are important for propofol actions ␣ ␤ ␥ 1 2 2 combinations being predominant in the adult mam- in both GABAAR and GlyRs. However, little is known about malian central nervous system.1,2 the contribution of other receptor regions. In this regard, a recent study has demonstrated that the large intracellular GABAAR and GlyR play important roles in the actions of 5–9 ␣ general anesthetics, including propofol, which is widely loop (LIL) of the 1 GlyR can influence the allosteric effects 29,30 used in intensive care units.10 Previous studies indicated that exerted by ethanol, which has been proposed to act at a these receptors contain sites important for propofol ac- site in the TM domains. Therefore, in the current study we 6,11,12 ␣ investigated the influence of the LIL on the allosteric action Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/115/3/464/254366/0000542-201109000-00010.pdf by guest on 02 October 2021 tion. For instance, residues in the TM domains in 1 ␤ of propofol in two members of the Cys-loop superfamily. and 2/3 subunits of the GABAAR were shown to be impor- tant for actions of anesthetics, including propofol.13–21 Ex- Our results identified a single phenylalanine residue, con- ␣ periments using a photoreactive analog of etomidate identi- served in the 1 subunit of both GABAARs and GlyRs, ␣ ␤ which affects their sensitivity to propofol. These results pro- fied two residues ( 1M236 in M1 and 2M286 in M3) as part of a binding pocket for this anesthetic.22 In addition, vide novel information about the relevance of the LIL in the allosteric modulation of the Cys-loop superfamily. based on the capacity of propofol to protect a sulfhydryl- specific reagent from reacting with a substituted cysteine, it was proposed that M286 in M3 served as an anesthetic bind- Materials and Methods ␤ 23 Complementary DNA Constructs. ing site in 2. A more recent study showed that binding of Mutations were inserted the photoreactive analog of etomidate to this site was either using the QuickChange site-directed mutagenesis kit (Strat- directly or allosterically inhibited by other general anesthet- agene, La Jolla, CA) in constructs encoding the human GlyR 24 ␣ ics, suggesting complex intramolecular interactions. In ad- 1 subunit subcloned in the pCI vector (Promega, Madison, ␣ ␤ dition to binding sites in TM2/TM3of ␣/␤ subunits in WI) and the rat 1 and 2 GABAAR subunits subcloned in the pRK5 vector (Clontech, Mountain View, CA). All mu- GABAAR, a tyrosine in TM4 (Y444) was found to influence the action of propofol, but not etomidate, on the receptor.14 tations were confirmed by full sequencing. The GlyRs and Studies in animal and molecular experimental models GABAAR amino acids were numbered according to their position in the mature protein sequence. have shown that the sites of general anesthetics on GABAAR and GlyR are somewhat overlapping for different chemical Cell Culture and Transfection. HEK293 cells (CRL-1573; structures. For example, transgenic mice carrying propofol- American Type Culture Collection, Manassas, VA) were cul- ␤ tured using standard methods. For the GlyR experiments, insensitive GABAARs ( 3N26 5M) also showed resistance to etomidate and exhibited substantial reductions in the mod- HEK293 cells were cotransfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with the ␣ GlyR plus the ulatory actions of the volatile anesthetic enflurane.19 Simi- 1 pGreenLantern plasmid (Invitrogen) codifying the green flu- larly, it was reported that residues S267 and A288 of ␣ 1 orescent protein (ratio 1:1; 2 ␮g DNA for each plasmid). GlyRs,25 which previously had been reported as critical for Expression of green fluorescent protein was used as a marker modulation by alcohols and enflurane,26 also affected propo- of positively transfected HEK293 cells and recordings were fol sensitivity. made after 18–36 h. In some experiments in which Although these previous studies have predicted that sev- ␣ ␤ ␥ GABAAR 1 2 2 subunits were expressed, HEK293 cells eral residues might constitute a propofol binding pocket, the were cotransfected using Lipofectamine 2000 (Invitrogen) absence of high-resolution structures of drug-receptor com- ␣ ␤ with the 1 and 2 subunits subcloned in the pRK5 vector plexes for eukaryotic receptors has hindered a complete un- ␥ and