Chapter Three - Allosteric Modulation of GABAA Receptors via Multiple Drug-Binding Sites

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Abstract

GABAA receptors are ligand-gated ion channels composed of five subunits that can be opened by GABA and be modulated by multiple pharmacologically and clinically important drugs. Over the time, hundreds of compounds from different structural classes have been demonstrated to modulate, directly activate, or inhibit GABAA receptors, and most of these compounds interact with more than one binding site at these receptors. Crystal structures of proteins and receptors homologous to GABAA receptors as well as homology modeling studies have provided insights into the possible location of ligand interaction sites. Some of these sites have been identified by mutagenesis, photolabeling, and docking studies. For most of these ligands, however, binding sites are not known. Due to the high flexibility of GABAA receptors and the existence of multiple drug-binding sites, the unequivocal identification of interaction sites for individual drugs is extremely difficult. The existence of multiple GABAA receptor subtypes with distinct subunit composition, the contribution of distinct subunit sequences to binding sites of different receptor subtypes, as well as the observation that even subunits not directly contributing to a binding site are able to influence affinity and efficacy of drugs, contribute to a unique pharmacology of each GABAA receptor subtype. Thus, each receptor subtype has to be investigated to identify a possible subtype selectivity of a compound. Although multiple binding sites make GABAA receptor pharmacology even more complicated, the exploitation of ligand interaction with novel-binding sites also offers additional possibilities for a subtype-selective modulation of GABAA receptors.

Introduction

GABAA receptors are the major inhibitory transmitter receptors in the brain. They are chloride ion channels that can be opened by GABA and are composed of five subunits. The existence of six α, three β, three γ, one δ, one ɛ, one θ, one π, and three ρ subunits and their distinct regional and cellular distribution in the brain gives rise to a multiplicity of GABAA receptor subtypes with different subunit composition and distinct pharmacological properties (Olsen & Sieghart, 2008). The majority of GABAA receptors, however, are composed of two α, two β, and one γ2 subunit. GABAA receptors are the site of action of a variety of pharmacologically and clinically important drugs such as benzodiazepines, barbiturates, neuroactive steroids, inhalation and intravenous anesthetics, and convulsants, which allosterically modulate GABA-induced currents via distinct binding sites (Sieghart, 1995). The presence of multiple allosteric binding sites at single GABAA receptors results in an extremely complex pharmacology of these receptors and raises the question where all these binding sites are located. Knowledge on the location and structure of these binding sites is essential for understanding GABAA receptor modulation by these clinically important drugs. In addition, this knowledge as well as that on homologous sites in other GABAA receptor subtypes is a prerequisite for a future structure-based drug design that could dramatically accelerate the development of novel subtype-selective drugs of potential therapeutic use (Rudolph and Knoflach, 2011, Rudolph and Mohler, 2014).

In the last couple of years, a variety of biochemical and molecular pharmacological techniques have been applied to identify and locate the various binding sites at GABAA receptors. These techniques include site-directed mutagenesis with functional or ligand-binding analysis, cysteine substitution and modifier accessibility with and without ligand protection, photoaffinity labeling, X-ray crystallography, and homology modeling. In this chapter, our current knowledge on the location and possible structure of various GABAA receptor-binding sites is summarized and the advantages and disadvantages of the various localization techniques are discussed. Although most of our knowledge has been gained by investigating α1β2/3γ2 receptors, the most abundant receptors in the brain, similar binding sites can also be found in most, if not all, GABAA receptors. Differences in homologous binding sites possibly can be used for a selective modulation of specific receptor subtypes.

Section snippets

Structure of GABAA Receptors

GABAA receptors are composed of five subunits that form the central chloride channel. Each subunit contains a large N-terminal extracellular domain, four transmembrane domains (TMs) each forming an α-helix, a large intracellular loop between TM3 and TM4, and a short extracellular C-terminus (Schofield et al., 1987). Experiments investigating the subunit arrangement of GABAA receptors composed of 2α, 2β, and one γ subunit have indicated that α and β subunits alternate with each other and are

GABA-Binding Sites

Over the years, considerable insights into the GABAA receptor orthosteric binding pocket have been obtained from site-directed mutagenesis studies, from studies using [3H]muscimol for photolabeling, or from receptor-binding studies using a range of GABA-site ligands (Petersen et al., 2013, Smith and Olsen, 1995). It is now clear that the GABA-binding site is located at the extracellular β+α − interface of GABAA receptors (Fig. 1A). Since the majority of GABAA receptors contain 2α, 2β, and one γ

Interaction of benzodiazepine-binding site ligands with the α+γ − interface of GABAA receptors

Benzodiazepines, such as chlordiazepoxide or diazepam, were introduced into clinical use in the 1960s and due to their anxiolytic, anticonvulsant, sedative hypnotic, and muscle relaxant properties soon became the most commonly prescribed drugs in therapeutic use. After the first cloning of GABAA receptor subunit cDNAs (Schofield et al., 1987), [3H]flunitrazepam-binding studies as well as studies investigating the electrophysiological effects of benzodiazepines at recombinant GABAA receptor

Picrotoxinin-Binding Sites

After the identification of GABAA receptors as being activated by GABA and inhibited by bicuculline (Curtis, Phillis, & Watkins, 1959), picrotoxin was identified as an additional noncompetitive inhibitor of these receptors (Curtis et al., 1969, Johnston, 1978). This inhibitor was not as selective for GABAA receptors as bicuculline and at higher concentrations also inhibited glycine receptors as well as other receptors from the same superfamily. The identification of high-affinity radioligands

Binding Sites for Anesthetics

Drugs acting via the benzodiazepine site at the extracellular α+γ − interface, or the pyrazoloquinolinones acting via the extracellular α+β − interface, can only modulate GABAA receptors but not directly open the channel in the absence of GABA. In contrast, barbiturates, neuroactive steroids, and anesthetics at low concentrations can enhance GABA-induced currents, whereas at higher concentrations, they are able to directly activate GABAA receptors in the absence of GABA. In addition,

Alcohol-binding sites in the transmembrane domain

Ethanol and longer chain alcohols exhibit a multiplicity of actions at various receptors and proteins. Nevertheless, the potencies of the n-alcohol series for physiologic immobilization and anesthesia parallel that for the potentiation of native GABA-induced currents (Nakahiro, Arakawa, Nishimura, & Narahashi, 1996), suggesting that GABAA receptors are an important site of action of alcohols. Over the time, several alcohol-binding sites have been identified in GABAA receptors, and depending on

Cannabinoid-Binding Site

The endocannabinoid system is part of a complex lipid signaling network involving the G protein-coupled receptors CB1 and CB2. Several lines of evidence indicate that endocannabinoids are also involved in the regulation of GABA and glutamate release (Rea, Roche, & Finn, 2007). In addition, recently, it was demonstrated that 2-arachidonyl glycerol and other endocannabinoids are able to directly modulate GABAA receptors via a novel-binding site located in the TM4 domain of GABAA receptors

Avermectin B1a-Binding Site

Avermectin B1a, an anthelmintic macrocyclic lactone, is widely used as an antiparasitic agent in domestic animals, usually as a mixture (ivermectin) of avermectin B1a and avermectin B1b. The target of its antiparasitic action is believed to be an ivermectin-sensitive glutamate-gated Cl channel that is directly activated by ivermectin at nanomolar concentrations and that is found exclusively in invertebrates (Lynagh & Lynch, 2012). At higher (micromolar) concentrations, avermectin B1a also

Binding Sites of Ions

In addition to the modulation by multiple drugs, GABAA receptors are also modulated by various cations, such as protons (Huang et al., 2004, Wilkins et al., 2002), Zn2 + (Fisher and Macdonald, 1998, Horenstein and Akabas, 1998, Hosie et al., 2003), La3 + (Zhu, Wang, Corsi, & Vicini, 1998), or Cu2 + (McGee, Houston, & Brickley, 2013). Again, multiple binding sites for most of these ions have been identified by site-directed mutagenesis that are located either within the ion channel or within the

Conclusion

From the previous chapters, it is clear that most, if not all, GABAA receptor ligands can interact with more than one binding site at these receptors. Most of these binding sites are not exactly defined yet and it can be assumed that identification of these sites will be an extremely difficult task given the high flexibility of the receptors and the existence of multiple binding sites. Since binding of a ligand in most cases causes changes in the structure of the receptor, a second ligand with

Conflict of Interest

The author has no conflicts of interest to declare.

References (138)

  • G. Drexler et al.

    Evidence for association of a high affinity avermectin binding site with the benzodiazepine receptor

    European Journal of Pharmacology

    (1984)
  • G. Drexler et al.

    Properties of a high affinity binding site for [3H]avermectin B1a

    European Journal of Pharmacology

    (1984)
  • B.E. Erkkila et al.

    Stoichiometric pore mutations of the GABAAR reveal a pattern of hydrogen bonding with picrotoxin

    Biophysical Journal

    (2008)
  • M. Ernst et al.

    Comparative modeling of GABAA receptors: Limits, insights, future developments

    Neuroscience

    (2003)
  • C.A. Hauser et al.

    Flunitrazepam has an inverse agonistic effect on recombinant alpha6beta2gamma2-GABAA receptors via a flunitrazepam-binding site

    The Journal of Biological Chemistry

    (1997)
  • A.M. Hosie et al.

    Neurosteroid binding sites on GABAA receptors

    Pharmacology & Therapeutics

    (2007)
  • S.S. Jayakar et al.

    Multiple propofol binding sites in a gamma-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog

    The Journal of Biological Chemistry

    (2014)
  • N. Karim et al.

    Low nanomolar GABA effects at extrasynaptic alpha4beta1/beta3delta GABAA receptor subtypes indicate a different binding mode for GABA at these receptors

    Biochemical Pharmacology

    (2012)
  • K.H. Kaur et al.

    Unanticipated structural and functional properties of delta-subunit-containing GABAA receptors

    The Journal of Biological Chemistry

    (2009)
  • H. Knust et al.

    The discovery and unique pharmacological profile of RO4938581 and RO4882224 as potent and selective GABAA alpha5 inverse agonists for the treatment of cognitive dysfunction

    Bioorganic & Medicinal Chemistry Letters

    (2009)
  • M.D. Krasowski et al.

    Methionine 286 in transmembrane domain 3 of the GABAA receptor beta subunit controls a binding cavity for propofol and other alkylphenol general anesthetics

    Neuropharmacology

    (2001)
  • G.D. Li et al.

    Numerous classes of general anesthetics inhibit etomidate binding to gamma-aminobutyric acid type A (GABAA) receptors

    The Journal of Biological Chemistry

    (2010)
  • T. Lynagh et al.

    Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

    The Journal of Biological Chemistry

    (2011)
  • M. Nakahiro et al.

    Potentiation of GABA-induced Cl current by a series of n-alcohols disappears at a cutoff point of a longer-chain n-alcohol in rat dorsal root ganglion neurons

    Neuroscience Letters

    (1996)
  • P. Perret et al.

    Interaction of non-competitive blockers within the gamma-aminobutyric acid type A chloride channel using chemically reactive probes as chemical sensors for cysteine mutants

    The Journal of Biological Chemistry

    (1999)
  • J. Ramerstorfer et al.

    The point mutation gamma 2F77I changes the potency and efficacy of benzodiazepine site ligands in different GABAA receptor subtypes

    European Journal of Pharmacology

    (2010)
  • D. Rusch et al.

    Gating allosterism at a single class of etomidate sites on alpha1beta2gamma2L GABAA receptors accounts for both direct activation and agonist modulation

    The Journal of Biological Chemistry

    (2004)
  • M. Bali et al.

    Defining the propofol binding site location on the GABAA receptor

    Molecular Pharmacology

    (2004)
  • S.W. Baumann et al.

    Individual properties of the two functional agonist sites in GABAA receptors

    The Journal of Neuroscience

    (2003)
  • R. Baur et al.

    Molecular analysis of the site for 2-arachidonylglycerol (2-AG) on the beta(2) subunit of GABAA receptors

    Journal of Neurochemistry

    (2013)
  • R. Baur et al.

    Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

    Journal of Neurochemistry

    (2008)
  • D. Belelli et al.

    The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid

    Proceedings of the National Academy of Sciences of the United States of America

    (1997)
  • R. Bergmann et al.

    A unified model of the GABAA receptor comprising agonist and benzodiazepine binding sites

    PLoS One

    (2013)
  • M.T. Bianchi et al.

    Neurosteroids shift partial agonist activation of GABAA receptor channels from low- to high-efficacy gating patterns

    The Journal of Neuroscience

    (2003)
  • N. Bocquet et al.

    X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation

    Nature

    (2009)
  • K. Brejc et al.

    Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors

    Nature

    (2001)
  • I.N. Cestari et al.

    The agonistic action of pentobarbital on GABAA beta-subunit homomeric receptors

    Neuroreport

    (1996)
  • L. Chen et al.

    Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site

    Proceedings of the National Academy of Sciences of the United States of America

    (2006)
  • Z.W. Chen et al.

    Neurosteroid analog photolabeling of a site in the third transmembrane domain of the beta3 subunit of the GABAA receptor

    Molecular Pharmacology

    (2012)
  • Z.W. Chen et al.

    GABAA receptor associated proteins: A key factor regulating GABAA receptor function

    Journal of Neurochemistry

    (2007)
  • D.C. Chiara et al.

    Mapping general anesthetic binding site(s) in human alpha1beta3 gamma-aminobutyric acid type A receptors with [(3)H]TDBzl-etomidate, a photoreactive etomidate analogue

    Biochemistry

    (2012)
  • D.C. Chiara et al.

    Photoaffinity labeling the propofol binding site in GLIC

    Biochemistry

    (2014)
  • D.R. Curtis et al.

    The depression of spinal neurones by gamma-amino-n-butyric acid and beta-alanine

    The Journal of Physiology

    (1959)
  • P.A. Davies et al.

    Modulation by general anaesthetics of rat GABAA receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in human embryonic kidney 293 cells

    British Journal of Pharmacology

    (1997)
  • C.D. Dellisanti et al.

    Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution

    Nature Neuroscience

    (2007)
  • R. Desai et al.

    Gamma-amino butyric acid type A receptor mutations at beta2N265 alter etomidate efficacy while preserving basal and agonist-dependent activity

    Anesthesiology

    (2009)
  • R. Dey et al.

    In search of allosteric modulators of a7-nAChR by solvent density guided virtual screening

    Journal of Biomolecular Structure & Dynamics

    (2011)
  • R. Dias et al.

    Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines

    The Journal of Neuroscience

    (2005)
  • G.H. Dillon et al.

    Enhancement by GABA of the association rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype

    British Journal of Pharmacology

    (1995)
  • I. Ducic et al.

    gamma-Aminobutyric acid gating of Cl channels in recombinant GABAA receptors

    The Journal of Pharmacology and Experimental Therapeutics

    (1995)
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