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