Skip to main content

Advertisement

SpringerLink
Log in

The general anesthetic etomidate and fenamate mefenamic acid oppositely affect GABAAR and GlyR: a structural explanation

  • Original Article
  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

GABA and glycine act as inhibitory neurotransmitters in the CNS. Inhibitory neurotransmission is mediated via activation of ionotropic GABAA and glycine receptors. We used a modeling approach to explain the opposite effects of the general anesthetic etomidate (ETM) and fenamate mefenamic acid (MFA) on GABA- and glycine-activated currents recorded in isolated cerebellar Purkinje cells and hippocampal pyramidal neurons, respectively. These drugs potentiated GABAARs but blocked GlyRs. We built a homology model of α1β GlyR based on the cryo-EM structure of open α1 GlyR, used the α1β3γ2 GABAAR structure from the PDB, and applied Monte-Carlo energy minimization to optimize models of receptors and ligand-receptor complexes. In silico docking suggests that ETM/MFA bind at the transmembrane β( +)/α( −) intersubunit interface in GABAAR. Our models predict that the bulky side chain of the highly conserved Arg19′ residue at the plus interface side wedges the interface and maintains the conducting receptor state. We hypothesized that MFA/ETM binding at the β( +)/α( −) interface leads to prolongation of receptor life-time in the open state. Having analyzed different GABAAR and GlyR structures available in the PDB, we found that mutual arrangement of the Arg19′ and Gln−26′ side chains at the plus and minus interface sides, respectively, plays an important role when the receptor switches from the open to closed state. We show that this process is accompanied by narrowing of the intersubunit interfaces, leading to extrusion of the Arg19′ side chain from the interface. Our models allow us to explain the lack of GlyR potentiation in our electrophysiological experiments.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Data availability

Data can be obtained by requesting the author.

References

  • Aroeira RI, Ribeiro JA, Sebastiao AM, Valente CA (2011) Age-related changes of glycine receptor at the rat hippocampus: from the embryo to the adult. J Neurochem 118:339–353

    CAS  PubMed  Google Scholar 

  • Bannwarth B, Netter P, Pourel J, Royer RJ, Gaucher A (1989) Clinical pharmacokinetics of nonsteroidal anti-inflammatory drugs in the cerebrospinal fluid. Biomed Pharmacother 43:121–126

    CAS  PubMed  Google Scholar 

  • Belelli D, Lambert JJ, Peters JA, Wafford K, Whiting PJ (1997) The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid. Proc Natl Acad Sci USA 94:11031–11036

    CAS  PubMed  Google Scholar 

  • Bode A, Lynch JW (2013) Analysis of hyperekplexia mutations identifies transmembrane domain rearrangements that mediate glycine receptor activation. J Biol Chem 288:33760–33771

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bode A, Lynch JW (2014) The impact of human hyperekplexia mutations on glycine receptor structure and function. Mol Brain 7:2

    PubMed  PubMed Central  Google Scholar 

  • Brooks CL, Pettitt BM, Karplus M (1985) Structural and energetic effects of truncating long ranged interactions in ionic polar fluids. J Chem Phys 83:5897–5908

    CAS  Google Scholar 

  • Cerdan AH, Martin NE, Cecchini M (2018) An Ion-permeable state of the glycine receptor captured by molecular dynamics. Structure 26:1555–1562.e1554

    CAS  PubMed  Google Scholar 

  • Connolly CN, Wafford KA (2004) The Cys-loop superfamily of ligand-gated ion channels: the impact of receptor structure on function. Biochem Soc Trans 32:529–534

    CAS  PubMed  Google Scholar 

  • Coyne L, Su J, Patten D, Halliwell RF (2007) Characterization of the interaction between fenamates and hippocampal neuron GABA(A) receptors. Neurochem Int 51:440–446

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cushman DW, Cheung HS (1976) Effect of substrate concentration on inhibition of prostaglandin synthetase of bull seminal vesicles by anti-inflammatory drugs and fenamic acid analogs. Biochem Biophys Acta 424:449–459

    CAS  PubMed  Google Scholar 

  • Damgen MA, Biggin PC (2020) A refined open state of the glycine receptor obtained via molecular dynamics simulations. Structure 28(130–139):e132

    Google Scholar 

  • Dewar MJS, Zoebisch EG, Healy EF, Stewart JJP (1985) AM1: a new general purpose quantum mechanical molecular model. J Am Chem Soc 107:3902–3909

    CAS  Google Scholar 

  • Du J, Lu W, Wu S, Cheng Y, Gouaux E (2015) Glycine receptor mechanism elucidated by electron cryo-microscopy. Nature 526:224–229

    CAS  PubMed  PubMed Central  Google Scholar 

  • Durisic N, Godin AG, Wever CM, Heyes CD, Lakadamyali M, Dent JA (2012) Stoichiometry of the human glycine receptor revealed by direct subunit counting. J Neurosci 32:12915–12920

    CAS  PubMed  PubMed Central  Google Scholar 

  • Estrada-Mondragon A, Lynch JW (2015) Functional characterization of Ivermectin binding sites in alpha1beta2gamma2L GABA(A) receptors. Front Mol Neurosci 8:55

    PubMed  PubMed Central  Google Scholar 

  • Forman SA (2011) Clinical and molecular pharmacology of etomidate. Anesthesiology 114:695–707

    CAS  PubMed  PubMed Central  Google Scholar 

  • Forman SA, Miller KW (2016) Mapping general anesthetic sites in heteromeric gamma-aminobutyric acid type A receptors reveals a potential for targeting receptor subtypes. Anesth Analg 123:1263–1273

    CAS  PubMed  PubMed Central  Google Scholar 

  • Galzi JL, Devillers-Thiery A, Hussy N, Bertrand S, Changeux JP, Bertrand D (1992) Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature 359:500–505

    CAS  PubMed  Google Scholar 

  • Garden DP, Zhorov BS (2010) Docking flexible ligands in proteins with a solvent exposure- and distance-dependent dielectric function. J Comput Aided Mol Des 24:91–105

    CAS  PubMed  Google Scholar 

  • Gonzalez-Gutierrez G, Wang Y, Cymes GD, Tajkhorshid E, Grosman C (2017) Chasing the open-state structure of pentameric ligand-gated ion channels. J Gen Physiol 149:1119–1138

    CAS  PubMed  PubMed Central  Google Scholar 

  • Grudzinska J, Schemm R, Haeger S, Nicke A, Schmalzing G, Betz H, Laube B (2005) The beta subunit determines the ligand binding properties of synaptic glycine receptors. Neuron 45:727–739

    CAS  PubMed  Google Scholar 

  • Halliwell RF, Thomas P, Patten D, James CH, Martinez-Torres A, Miledi R, Smart TG (1999) Subunit-selective modulation of GABAA receptors by the non-steroidal anti-inflammatory agent, mefenamic acid. Eur J Neurosci 11:2897–2905

    CAS  PubMed  Google Scholar 

  • Hevers W, Luddens H (1998) The diversity of GABAA receptors. Pharmacological and electrophysiological properties of GABAA channel subtypes. Mol Neurobiol 18:35–86

    CAS  PubMed  Google Scholar 

  • Hibbs RE, Gouaux E (2011) Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474:54–60

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hill-Venning C, Belelli D, Peters JA, Lambert JD (1997) Subunit-dependent interaction of the general anaesthetic etomidate with the g-aminobutyric acid type A receptor. Br J Pharm 120:749–756

    CAS  Google Scholar 

  • Itier V, Depoortere H, Scatton B, Avenet P (1996) Zolpidem functionally discriminates subtypes of native GABAA receptors in acutely dissociated rat striatal and cerebellar neurons. Neuropharmacology 35:137–145

    CAS  PubMed  Google Scholar 

  • Jonsson S, Morud J, Pickering C, Adermark L, Ericson M, Soderpalm B (2012) Changes in glycine receptor subunit expression in forebrain regions of the Wistar rat over development. Brain Res 1446:12–21

    CAS  PubMed  Google Scholar 

  • Jurd R, Arras M, Lambert S, Drexler B, Siegwart R, Crestani F, Zaugg M, Vogt KE, Ledermann B, Antkowiak B, Rudolph U (2003) General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. FASEB J 17:250–252

    CAS  PubMed  Google Scholar 

  • Keramidas A, Moorhouse AJ, Schofield PR, Barry PH (2004) Ligand-gated ion channels: mechanisms underlying ion selectivity. Prog Biophys Mol Biol 86:161–204

    CAS  PubMed  Google Scholar 

  • Kumar A, Basak S, Rao S, Gicheru Y, Mayer ML, Sansom MSP, Chakrapani S (2020) Mechanisms of activation and desensitization of full-length glycine receptor in lipid nanodiscs. Nat Commun 11:3752

    CAS  PubMed  PubMed Central  Google Scholar 

  • Laurie DJ, Seeburg PH, Wisden W (1992) The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J Neurosci 12:1063–1076

    CAS  PubMed  PubMed Central  Google Scholar 

  • Laverty D, Desai R, Uchanski T, Masiulis S, Stec WJ, Malinauskas T, Zivanov J, Pardon E, Steyaert J, Miller KW, Aricescu AR (2019) Cryo-EM structure of the human alpha1beta3gamma2 GABAA receptor in a lipid bilayer. Nature 565:516–520

    CAS  PubMed  Google Scholar 

  • Legendre P (2001) The glycinergic inhibitory synapse. Cell Mol Life Sci 58:760–793

    CAS  PubMed  Google Scholar 

  • Li GD, Chiara DC, Sawyer GW, Husain SS, Olsen RW, Cohen JB (2006) Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. J Neurosci 26:11599–11605

    CAS  PubMed  PubMed Central  Google Scholar 

  • Li Z, Scheraga HA (1987) Monte Carlo-minimization approach to the multiple-minima problem in protein folding. Proc Natl Acad Sci USA 84:6611–6615

    CAS  PubMed  Google Scholar 

  • Low SE, Ito D, Hirata H (2018) Characterization of the zebrafish glycine receptor family reveals insights into glycine receptor structure function and stoichiometry. Fron Mol Neurosci 11:286

    CAS  Google Scholar 

  • Lynch JW (2009) Native glycine receptor subtypes and their physiological roles. Neuropharmacology 56:303–309

    CAS  PubMed  Google Scholar 

  • Maleeva G, Peiretti F, Zhorov BS, Bregestovski P (2017) Voltage-dependent inhibition of glycine receptor channels by niflumic acid. Front Mol Neurosci 10:125

    PubMed  PubMed Central  Google Scholar 

  • Masiulis S, Desai R, Uchanski T, Serna Martin I, Laverty D, Karia D, Malinauskas T, Zivanov J, Pardon E, Kotecha A, Steyaert J, Miller KW, Aricescu AR (2019) GABAA receptor signalling mechanisms revealed by structural pharmacology. Nature 565:454–459

    CAS  PubMed  PubMed Central  Google Scholar 

  • Miller C (1989) Genetic manipulation of ion channels: a new approach to structure and mechanism. Neuron 2:1195–1205

    CAS  PubMed  Google Scholar 

  • Miller PS, Smart TG (2010) Binding, activation and modulation of Cys-loop receptors. Trends Pharmacol Sci 31:161–174

    CAS  PubMed  Google Scholar 

  • Olsen RW (2018) GABAA receptor: positive and negative allosteric modulators. Neuropharmacology 136:10–22

    CAS  PubMed  PubMed Central  Google Scholar 

  • Patrizio A, Renner M, Pizzarelli R, Triller A, Specht CG (2017) Alpha subunit-dependent glycine receptor clustering and regulation of synaptic receptor numbers. Sci Rep 7:10899

    CAS  PubMed  PubMed Central  Google Scholar 

  • Patten D, Foxon GR, Martin KF, Halliwell RF (2001) An electrophysiological study of the effects of propofol on native neuronal ligand-gated ion channels. Clin Exp Pharmacol Physiol 28:451–458

    CAS  PubMed  Google Scholar 

  • Pistis M, Belelli D, Peters JA, Lambert JJ (1997) The interaction of general anaesthetics with recombinant GABAA and glycine receptors expressed in Xenopus laevis oocytes: a comparative study. Br J Pharmacol 122:1707–1719

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rossokhin A (2019) Multidirectional action of fenamates and general anesthetics on GABAA and glycine receptors structural explanation. FEBS Open Bio 9(S1):181

    Google Scholar 

  • Rossokhin A, Dreker T, Grissmer S, Zhorov BS (2011) Why does the inner-helix mutation A413C double the stoichiometry of Kv1.3 channel block by emopamil but not by verapamil? Mol Pharmacol 79:681–691

    CAS  PubMed  Google Scholar 

  • Rossokhin AV (2017) Homology modeling of the transmembrane domain of the GABAA receptor. Biophysics 62:708–716

    CAS  Google Scholar 

  • Rossokhin AV, Sharonova IN, Bukanova JV, Kolbaev SN, Skrebitsky VG (2014) Block of GABA receptor ion channel by penicillin: electrophysiological and modeling insights toward the mechanism. Mol Cell Neurosci 63:72–82

    CAS  PubMed  Google Scholar 

  • Rossokhin AV, Sharonova IN, Dvorzhak A, Bukanova JV, Skrebitsky VG (2019) The mechanisms of potentiation and inhibition of GABAA receptors by non-steroidal anti-inflammatory drugs, mefenamic and niflumic acids. Neuropharmacology 160:107795

    CAS  PubMed  Google Scholar 

  • Rossokhin AV, Zhorov BS (2016) Side chain flexibility and the pore dimensions in the GABAA receptor. J Comput Aided Mol Des 30:559–567

    CAS  PubMed  Google Scholar 

  • Sauguet L, Howard RJ, Malherbe L, Lee US, Corringer PJ, Harris RA, Delarue M (2013) Structural basis for potentiation by alcohols and anaesthetics in a ligand-gated ion channel. Nat Commun 4:1697

    PubMed  PubMed Central  Google Scholar 

  • Scott S, Lynch JW, Keramidas A (2015) Correlating structural and energetic changes in glycine receptor activation. J Biol Chem 290:5621–5634

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sieghart W (2006) Structure, pharmacology, and function of GABAA receptor subtypes. Adv Pharmacol 54:231–263

    CAS  PubMed  Google Scholar 

  • Sieghart W (2015) Allosteric modulation of GABAA receptors via multiple drug-binding sites. Adv Pharmacol 72:53–96

    CAS  PubMed  Google Scholar 

  • Siegwart R, Jurd R, Rudolph U (2002) Molecular determinants for the action of general anesthetics at recombinant alpha2beta3gamma2 gamma-aminobutyric acid A receptors. J Neurochem 80:140–148

    CAS  PubMed  Google Scholar 

  • Smith AJ, Oxley B, Malpas S, Pillai GV, Simpson PB (2004) Compounds exhibiting selective efficacy for different beta subunits of human recombinant gamma-aminobutyric acid A receptors. J Pharmacol Exp Ther 311:601–609

    CAS  PubMed  Google Scholar 

  • Stewart DS, Hotta M, Li GD, Desai R, Chiara DC, Olsen RW, Forman SA (2013) Cysteine substitutions define etomidate binding and gating linkages in the alpha-M1 domain of gamma-aminobutyric acid type A (GABAA) receptors. J Biol Chem 288:30373–30386

    CAS  PubMed  PubMed Central  Google Scholar 

  • Trick JL, Chelvaniththilan S, Klesse G, Aryal P, Wallace EJ, Tucker SJ, Sansom MSP (2016) Functional annotation of ion channel structures by molecular simulation. Structure 24:2207–2216

    CAS  PubMed  PubMed Central  Google Scholar 

  • Webb TI, Lynch JW (2007) Molecular pharmacology of the glycine receptor chloride channel. Curr Pharm Des 13:2350–2367

    CAS  PubMed  Google Scholar 

  • Weiner J, Kollman PA, Case DA, Singh UC, Chio C, Alagona G, Profeta S, Weiner PK (1984) A new force field for molecular mechanical simulation of nucleic acids and proteins. J Am Chem Soc 106:765–784

    CAS  Google Scholar 

  • Woodward RM, Polenzani L, Miledi R (1994) Effects of fenamates and other nonsteroidal anti-inflammatory drugs on rat brain GABAA receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 268:806–817

    CAS  PubMed  Google Scholar 

  • Yang Z, Taran E, Webb TI, Lynch JW (2012) Stoichiometry and subunit arrangement of alpha1beta glycine receptors as determined by atomic force microscopy. Biochemistry 51:5229–5231

    CAS  PubMed  Google Scholar 

  • Zhang Z-X, Lü H, Dong X-P, Liu J, Xu T-L (2002) Kinetics of etomidate actions on GABAA receptors in the rat spinal dorsal horn neurons. Brain Res 953:93–100

    CAS  PubMed  Google Scholar 

  • Zhorov BS (1981) Vector method for calculating derivatives of energy of atom-atom interactions of complex molecules according to generalized coordinates. J Struct Chem 22:4–8

    Google Scholar 

Download references

Acknowledgements

In silico modeling of the GABAARs was performed as part of the Federal Project No. AAAA-A19-119091690081-1 and in silico modeling of the GlyRs was supported by the RFBR grant 18-015-00038. The research was carried out using the equipment of the shared research facilities of HPC computing resources at the Lomonosov Moscow State University.

Funding

In silico modeling of the GABAARs was performed as part of the Federal Project no. AAAA-A19-119091690081-1 and in silico modeling of the GlyRs was supported by the RFBR grant 18-015-00038.

Author information

Authors and Affiliations

  1. Research Center of Neurology, Moscow, Russia

    Alexey Rossokhin

Authors
  1. Alexey Rossokhin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alexey Rossokhin.

Ethics declarations

Conflicts of interest

There are no conflicts of interest or competing interests to be declared.

Ethical approval

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rossokhin, A. The general anesthetic etomidate and fenamate mefenamic acid oppositely affect GABAAR and GlyR: a structural explanation. Eur Biophys J 49, 591–607 (2020). https://doi.org/10.1007/s00249-020-01464-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00249-020-01464-7

Keywords

Search

Navigation