The GABAA receptor alpha+beta- interface: a novel target for subtype selective drugs
Doctoral thesis at the Medical University of Vienna
for obtaining the academic degree
Doctor of Philosophy
Submitted by
Mag.rer.nat. Joachim RAMERSTORFER
Supervisor:
Prof., Dr. Werner Sieghart
Center for Brain Research, Medical University Vienna,
Spitalgasse 4, 1090 Vienna
Vienna, 01/2012 Index
1 ABSTRACT ...... 4
2 ZUSAMMENFASSUNG ...... 6
3 INTRODUCTION ...... 8
3.1 Cys-loop receptor superfamily ...... 9
3.2 GABAA receptor structure and heterogeneity ...... 12
3.3 GABAA receptor pharmacology...... 14
3.4 Aim of this thesis ...... 17
4 MATERIALS AND METHODS ...... 19
4.1 Chemicals ...... 19
4.2 GABAA receptor subunits and point mutations ...... 19
4.3 Two electrode voltage clamp: ...... 20
4.3.1 In vitro transcription of capped cRNA ...... 20
4.3.1.1 Linearizing cDNA vectors ...... 20
4.3.1.2 Proteinase K-digestion ...... 21
4.3.1.3 Extraction of cDNA...... 21
4.3.1.4 Precipitation of cDNA by ethanol ...... 21
4.3.1.5 Transcription ...... 21
4.3.1.6 Extraction and Precipitation of RNA ...... 22
4.3.1.7 Polyadenylation ...... 22
4.3.1.8 Extraction and precipitation of mRNA ...... 22
4.3.2 Protein expression in Xenopus leavis oocytes ...... 23
4.3.3 Electrophysiological recordings ...... 24
5 RESULTS ...... 25
2
Index
5.1 The pyrazoloquinoline CGS 9895 enhances GABA-induced chloride flux in 13 receptors ...... 25
5.2 CGS 9895 is an antagonist at the benzodiazepine binding site .. 27
5.3 A steric hindrance approach indicates that CGS 9895 does not mediate its action via the +- interface ...... 30
5.4 Identification of suitable mutations for steric hindrance at the +- interface ...... 33
5.5 Application of the steric hindrance approach using two suitable point mutations at the +- interface ...... 39
5.6 CGS 9895 differentially modulates different receptor subtypes via its binding site at the +- interface ...... 47
6 DISCUSSION ...... 50
6.1 Steric hindrance – a novel tool for the identification of drug binding sites ...... 50
6.2 The anxiolytic actions of CGS 9895 seem to be mediated via the
benzodiazepine binding site of GABAA receptors containing 3 subunits...... 52
6.3 Drugs interacting with the +- interface are candidates for a
novel GABAA receptor pharmacology ...... 53
7 ABBREVIATIONS ...... 56
8 REFERENCES ...... 58
9 ACKNOWLEDGEMENT ...... 63
10 PUBLICATIONS ...... 64
11 CURRICULUM VITAE ...... 65
3
Abstract
1 ABSTRACT
GABAA receptors are the major inhibitory neurotransmitter receptors in the central nervous system and mediate fast synaptic inhibition by opening an intrinsic chloride ion channel upon GABA binding. The receptors contain binding sites for a number of pharmacologically important drugs which modulate GABA induced chloride flux by interacting with separate and distinct allosteric binding sites. So far, in addition to the two GABA binding sites, only the binding site for benzodiazepines, that is located at the interface formed by the plus side of the alpha (+) and the minus side of the gamma (-) subunit, has been unequivocally identified at the extracellular domain of these receptors. Many of the drugs showing effects at GABAA receptors, however, act via binding sites different from that of the benzodiazepines. One potential site of compound action at the extracellular domain of the receptor is the interface formed by the plus side of the alpha (+) and the minus side of the beta (-) subunit. This interface shares common properties with the benzodiazepine binding site, namely the + side. In a screening of 95 benzodiazepine site ligands, we identified the pyrazoloquinoline CGS 9895 as positive allosteric modulator at 13 receptors. CGS 9895 was able to stimulate 13 receptors to the same extent as 132 receptors, indicating that it does not require the benzodiazepine binding site for stimulation of receptors. Other experiments indicated that CGS 9895, in addition to its positive allosteric modulation, acts as allosteric antagonist at the benzodiazepine binding site of 132 receptors. To investigate whether CGS 9895 elicits its positive modulation via the +- interface we made use of a so-called steric hindrance approach: For that, single amino acid residues at the 1+ and the 3- side were mutated to cysteines and these cysteines were then covalently labelled by the cysteine reactive reagent, methanethiosulfonate ethylamine-biotin (MTSEA-biotin). Inhibition of the CGS 9895 effect then indicated that this effect was mediated via interaction with the 1+3- interface. In addition, we demonstrated that the intensity of the CGS 9895 effect was dependent on the types of and subunits present in the receptor. This suggested that CGS 9895 represents a prototype of drugs Abstract eliciting benzodiazepine like effects by interaction with the +- interface of
GABAA receptors. Since the +- interface is present in all heteropentameric
GABAA receptors, the action of these drugs will be much broader than that of benzodiazepines, that only interact with receptors. These drugs might thus become of clinical importance for the treatment of epilepsy.
Parts of this thesis have been published in:
Journal of Neuroscience, 2011, Jan 19; 31(3): 870 - 877
5
Abstract
2 ZUSAMMENFASSUNG
GABAA Rezeptoren sind die bedeutendsten inhibitorischen Neurotransmitter Rezeptoren des zentralen Nervensystems. Durch die Bindung von GABA kommt es zur Öffnung des intrinsischen Chlorid Ionen Kanals, was zur Übertragung von schneller synaptischer Inhibierung führt. Der Rezeptor hat Bindungsstellen für eine große Anzahl von pharmakologisch wichtigen Substanzen, welche GABA induzierte Chloridströme modulieren können. Dies geschieht durch die Interaktion mit spezifischen und separaten Bindungsstellen am GABAA Rezeptor. Bis jetzt wurde an der extrazellulären Domäne des Rezeptors, neben den zwei Bindungsstellen für GABA, nur die Bindungsstelle für Benzodiazepine eindeutig identifiziert. Die Benzodiazepin-Bindungsstelle befindet sich an der Schnittstelle der plus Seite der Alpha (+) und der minus
Seite der Gamma (-) Untereinheit. Der Großteil der Substanzen die am GABAA Rezeptor aktiv sind, bindet jedoch an anderen Stellen als der Benzodiazepin- Bindungsstelle. Eine mögliche weitere Substanzbindungsstelle an der extrazellulären Domäne des Rezeptors könnte an der Schnittstelle zwischen der plus Seite der Alpha (+) und der minus Seite der Beta (-) Untereinheit existieren. Diese Schnittstelle hat strukturelle Gemeinsamkeiten mit der Benzodiazepin-Bindungsstelle, nämlich die + Seite. In einer Untersuchung von 95 Substanzen, die mit hoher Affinität an die Benzodiazepin-Bindungsstelle binden, konnten wir das Pyrazoloquinolin CGS 9895 als positiven-allosterischen Modulator von 13 Rezeptoren identifizieren. Da diese Rezeptoren keine +-, sehr wohl aber +- Bindungsstellen besitzen, war der Effekt von der Benzodiazepin-Bindungsstelle unabhängig. Außerdem war CGS 9895 in der Lage 13 Rezeptoren und 132 Rezeptoren gleichermaßen zu stimulieren. Andere Untersuchungen wiesen darauf hin, dass CGS 9895 an der Benzodiazepin-Bindungsstelle von 132 Rezeptoren als allosterischer Antagonist wirkt, und somit über diese Stelle keine Wirkung auf den Rezeptor auslöst. Um die mögliche Bindungsstelle von CGS 9895 an der +- Schnittstelle zu identifizieren, benutzten wir einen sogenannten „Steric- hindrance“ Ansatz. Dabei wurden einzelne Aminosäuren an der + oder/und - 6
Abstract
Seite in Cysteine mutiert, um sie mit einem Cystein-reaktiven Reagenz, Methanthiosulfanat Ethylamin-Biotin (MTSEA-biotin) kovalent zu markieren. Die anschließend beobachtete Reduktion des CGS 9895 Effekts wies darauf hin, dass der positive allosterische Effekt über die +- Bindungsstelle ausgelöst wurde. Weiters konnten wir zeigen, dass die Stärke des Effekts von CGS 9895 von den unterschiedlichen Typen der - und -Untereinheiten im Rezeptor abhängig ist. CGS 9895 repräsentiert somit einen Prototypen von Substanzen die ähnliche Effekte wie Benzodiazepine haben, obwohl sie an die +-
Schnittstelle des GABAA Rezeptor binden. Da diese +- Schnittstelle, im
Gegensatz zur +- Schnittstelle, in allen heteropentameren GABAA Rezeptoren präsent ist, werden solche Substanzen eine weitaus breitere Wirkung als die Benzodiazepine haben. Diese Substanzen könnten somit auch gute Antikovulsiva sein und für die klinische Behandlung von Epilepsien Verwendung finden .
7
Introduction
3 INTRODUCTION
Accurate processing of information in an intact brain requires precisely concerted neuronal activity. Signalling is initiated by excitatory neurotransmitters, predominantly glutamate, and is regulated by inhibitory neurotransmitters, such as glycine and -aminobutyric acid (GABA).
GABA is the quantitatively most abundant inhibitory neurotransmitter in the central nervous system and elicits most of its actions via binding to GABAA receptors (Sieghart 1995). GABA is synthesized from glutamate, in a reaction catalysed by glutamic acid decarboxylase. GABA is stored pre-synaptically in vesicles and released into the synaptic cleft upon depolarization of the presynaptic membrane and binds to GABAA receptors at the post-synaptic terminal. After the release of GABA its concentration rises for a short time up to 3mM (Glykys and Mody 2007) within the synaptic cleft. GABA is then removed rapidly by GABA transporters at the pre-synaptic terminal or at the surrounding glia cells and is then either recycled into pre-synaptic vesicles or metabolized by GABA-transaminases (Minelli, Brecha et al. 1995).
There are two types of GABA receptors: the ionotropic GABAA receptors and the metabotropic GABAB receptors.
GABAA receptors are activated by the endogenous ligand GABA, as well as by muscimol and isoguvacine. They are competitively inhibited by bicuculline and - non-competitively by picrotoxin (Sieghart 1995). GABAA receptors are Cl ion channels which are activated upon GABA binding. In the adult brain in most cases there is a lower chloride concentration inside of neurons. Thus, opening of the channels leads to an influx of Cl- ions into the cell. This results in a fast decrease of the membrane potential and a reduced excitability of the cell (Sivilotti and Nistri 1991). In early development, in some neurons in the adult brain, or in the course of a disease state, the Cl- concentration within the cell can be higher, and opening of Cl- channels can thus cause an efflux of Cl- and thus, a depolarization (Ben-Ari 2002).
8
Introduction
GABAB receptor are activated by GABA and (-) baclofen and inhibited by phaclofen (Bowery 1993). These receptors, in contrast to GABAA receptors, are hetero-tetrameric G-protein-coupled receptors composed of GABAB1 and
GABAB2 subunits each consisting of seven membrane spanning domains (Pagano, Rovelli et al. 2001).
A third class of GABA receptors can also be found in the literature, the GABAC receptors. These are GABAA receptors composed of rho () subunits (Olsen and Sieghart 2008). Since these receptors are structurally related to GABAA receptors, they are now classified as GABAA receptor subtypes (Barnard et al., 1998; Olsen and Sieghart, 2008).
3.1 Cys-loop receptor superfamily
The GABAA receptor is part of the so-called cys-loop pentameric ligand- gated ion channel family (LGIC). Among this family are cation-selective receptors, such as the nicotinic acetylcholine receptor (nAChR), the ionotropic 2+ serotonin type 3 receptor (5HT3R) and a Zn -activated ion channel. The group of anion-selective receptors consists of the glycine receptor (GlyR) and the
GABAA receptor (Olsen and Sieghart 2008). All receptors of the LGIC family consist of five subunits, that are membrane spanning proteins and assemble circularly by forming a central pore through the membrane that is selective for the respective ions. Each of the five subunits, forming the receptor, have a long extracellular N-terminal domain, four trans-membrane (TM) domains, a short and a long intracellular loop connecting TM1 and TM2, or TM3 and TM4, respectively, a short extracellular loop between TM2 and TM3, and a short extracellular C-terminal domain (Fig. 1). The TM2 of the five subunits form the pore (Olsen and Sieghart 2008).
9
Introduction
Fig. 1 Topology of a single subunit of the cys-loop superfamily (Jacob, Moss et al. 2008).
The first information on the structure of LGIC has been obtained in 2001 (Brejc, van Dijk et al. 2001) from the crystal structure of a soluble homologue of the nAChR, the acetylcholine binding protein (AChBP). In 2005, the structure of nAChR of Torpedo marmorata has been revealed by the use of cryo-electron microscopy combined with image reconstruction at a resolution of 4 Å (Unwin 2005). These findings, together with sequence data, allowed the generation of homology models of the extracellular domain of the GABAA receptor (Ernst, Brauchart et al. 2003; Ernst, Bruckner et al. 2005). Such models for the first time revealed a more detailed picture of the structural characteristics of GABAA receptors and of their binding pockets. At the extracellular domain of the five subunits five interfaces are formed by neighbouring subunits at which five pockets are located (Fig. 2A). At the two / interfaces the GABA binding sites are located and at the / interface the benzodiazepine binding site is located. The binding sites at the interfaces are formed by so-called “loops”. The loops at the plus (principle) side are the loops A, B and C, the loops at the minus (complementary) side are the loops D, E and F that actually represent -strands (Ernst, Brauchart et al. 2003). The GABA binding sites are formed by the plus (+) side of the and the minus (-) side of the subunit, whereas the benzodiazepine binding site is formed by the plus (+) side of the and the minus (-) side of the subunit. The whole family of LGIC shares amino acid residues homologous to those in the GABA binding site, and these are as well 10
Introduction homologous to those in the benzodiazepine binding site (Ernst, Brauchart et al. 2003). The homology model also showed a number of solvent accessible cavities located at the trans-membrane domain of the GABAA receptor, in addition to the ones at the extracellular domain (Fig. 2B) (Ernst, Bruckner et al. 2005). The function of those cavities at the trans-membrane domain is still not clear, they might be essential for the movement of the receptor at different conformational stages, or they might serve as well as additional drug binding sites. The large number of putative drug binding sites could explain the complex pharmacology of the GABAA receptor (Ernst, Bruckner et al. 2005).
Fig. 2 Schematic drawing of a GABAA receptor consisting of 2 , 2 and one subunit, showing possible solvent accessible cavities (grey). (A) At the extracellular domain of the receptor, solvent accessible cavities are depicted as grey ellipses labelled with Latin numbers. These cavities are located at the interfaces formed by the “+”-side of one subunit and the “-“-side of the adjacent subunit. The two cavities labelled with 1 are the GABA binding sites, cavity labelled with 2 is the benzodiazepine binding site. For the cavities 3 and 4 no binding partners have been identified so far. (B) Transmembrane domain of the GABAA receptor. Elliptical cavities 1a, 2a, 3a and 4a are located at the respective interfaces below the extracellular domain. The circular cavities labelled with 5, 6 and 7 are located within the four transmembrane domains of the different subunits, labelled with Roman numbers. Cavity number 8 shows the ion- pore at the centre of the receptor.
11
Introduction
3.2 GABAA receptor structure and heterogeneity
GABAA receptors consists of five subunits that can belong to different subunit classes (Nayeem, Green et al. 1994) (Tretter, Ehya et al. 1997). 19 genes for mammalian GABAA receptor subunits have been identified (Simon, Wakimoto et al. 2004) these include six , three , three , one one , one , one and three . Theoretically, a vast variety of receptor subtypes could be built out of these different subunits, and this variety is further enhanced by the existence of additional splice variants of the 2 subunit, 2L (large) and 2S (small) (Whiting, McKernan et al. 1990; Kofuji, Wang et al. 1991). But so far, only a few of these possible subunit combinations have been clearly identified (Olsen and Sieghart 2008). As shown in Fig. 3, the most abundant type of
GABAA receptors is formed of two , two and one subunit (Barnard, Skolnick et al. 1998).
Fig. 3 GABAA receptor structure modified from (Olsen and Sieghart 2008).
GABAA receptor subunits are ubiquitously expressed all over the central nervous system and a variety of subunits can be co-expressed within the same neuron (Fritschy, Benke et al. 1992); (Wisden, Laurie et al. 1992).
Among the subunits, the 1 subunit is the most abundant one and it prevalently colocalizes with 2 and 2 subunits (Sieghart and Sperk 2002). The
12
Introduction
knock-out of the 1 subunit in mice leads to a 50% reduction of all GABAA receptors (Sur, Wafford et al. 2001) indicating the importance of this subunit. Compared to the 1 subunit, the 2 and the 3 subunits show a lower expression level in the brain, and the 5 subunit has an even lower expression in the whole brain, but is highly expressed in hippocampus (Pirker, Schwarzer et al. 2000), (Sieghart and Sperk 2002). The 4 and 6 subunits show a rather distinct expression in the forebrain and cerebellum, respectively.
Among the subunits, the 2 shows the broadest distribution with a very high expression level, comparable to the 1 subunit. As for the 1 subunit, it has been shown that a knock-out of the 2 subunit leads to 50% loss of GABAA receptors (Sur, Wafford et al. 2001). The 1 subunit shows the lowest expression of the three subunits and the 3 subunit has an expression level somewhat in between 2 and 1, but with a more distinct expression pattern.
Among the subunits, the 2 has the broadest distribution and highest expression level in the brain. Furthermore, it is the most frequently occurring subunit of all subunits and is contained in up to 75% of all GABAA receptors (Sieghart and Ernst 2005). The 2 subunit is prevalently co-expressed with and subunits (Olsen and Sieghart 2008). The other subunits, 1 and 3 show a rather minor expression level compared to 2.
The other subunits are considered, compared to the ones mentioned above, as rather subsidiary in respect of their expression levels and distribution.
Among those, the subunits are the most abundant. The subunit seems to be able to replace the subunit and builds receptors (Barrera, Betts et al. 2008). It is preferentially co-expressed with 4 and 6 together with subunits. Together with the 4 subunit it is particularly found in forebrain and cortical areas, and it is essential for the co-expression with 6 subunits in cerebellum.(Sieghart and Sperk 2002). In addition, the subunit is exclusively found extra- or perisynaptically (Nusser, Sieghart et al. 1998).
The subunit seems to be scarcely expressed and seems to combine with and subunits, but receptors containing this subunit are not yet well
13
Introduction characterized. The and subunits are so far even less well characterized (Korpi, Grunder et al. 2002), (Sieghart and Sperk 2002).
For synaptic expression, GABAA receptors require a 2 subunit to be incorporated together with 1, 2, or 3 subunits and a subunit. But 2 subunits are not exclusively expressed synaptically. Due to a far larger extrasynaptic as compared with the synaptic surface, 2 subunits actually have a higher extrasynaptic expression than a synaptic one (Kasugai, Swinny et al. 2010). Receptors consisting of 52 subunits, or a 4 or 6 subunit together with subunits seem to be located primarily extrasynaptically. Receptors containing a or an subunit are assumed to be solely extrasynaptic (Mody and Pearce 2004); (Semyanov, Walker et al. 2004); (Farrant and Nusser 2005).
3.3 GABAA receptor pharmacology
GABAA receptors are the site of action of a large variety of compounds which are able to modulate the GABA-mediated effect due to binding to separate and distinct allosteric binding sites (Sieghart 1995). Among those, many compounds are important for clinical and pharmacological applications, such as benzodiazepines, barbiturates, neuro-steroids, anaesthetics and convulsants (Sieghart 1995). So far, in addition to the GABA binding site, only the high affinity binding site for benzodiazepines has been unequivocally identified at the extracellular domain of the GABAA receptor (Fig. 2A and 4A). The binding of barbiturates has been reported to be dependent on the subunit type and to be influenced by the subunit (Korpi, Grunder et al. 2002; D'Hulst, Atack et al. 2009), but to be independent of the subunit. It seems that barbiturates have at least two different binding sites, both located in cavities of the trans-membrane domain of the receptor (Fig. 2B). Similarly neuro-steroids, volatile- and intravenous anaesthetics seem to have binding sites within the trans-membrane domain of the GABAA receptor (Fig. 2B). Neuro-steroids seem to have one putative binding site at the +- interface (Fig. 2B-1a) and one at the cavity inside the helical domain of the subunit (Fig. 2B -5) (Hosie, Wilkins et al. 2006; Hosie, Clarke et al. 2009). Volatile anaesthetics might share the
14
Introduction same cavity within the helical domain of the subunit just as the neuro-steroids (Fig. 2B-5) and have an additional putative binding site at the similar cavity at the subunit of the trans-membrane domain (Fig. 2B-6) (Ernst, Bruckner et al. 2005). Ligands at the benzodiazepine binding site can be classified depending on their effects at GABAA receptors. First there is the group of agonists, or positive allosteric modulators, such as the classical benzodiazepines. These compounds are able to enhance an ongoing GABA-induced current and have anxiolytic, anticonvulsant, muscle relaxant and sedative effects. The second group are the inverse agonists, or negative allosteric modulators. These compounds reduce an ongoing GABA-induced current and lead to convulsant, anxiogenic or stimulating effects. The third group is represented by the antagonists. These compounds bind to the benzodiazepine binding site but do not change or affect GABA-induced currents. But since they are able to displace agonists or inverse agonists from their binding site, they reduce or completely inhibit the action of these drugs (Sieghart 1995).
15
Introduction
Fig. 4 Top view onto the extracellular domain of GABAA receptors. Each subunit has assigned a plus (+)-and a minus (-)-side. Binding sites for GABA (GABA) are located at the interfaces formed by the (+)-side of the and the (-) - side of the subunits. (A) 132 receptors composed of one 2, two 1 and two 3 subunits. The binding site for benzodiazepine ligands (Bz) is located at the interface formed by the (+)-side of the and the (-) -side of the subunit. The interface of interest (?) is formed by the (+)-side of the and the (-) -side of the subunit. (B)13 receptors composed of two 1 and three 3 subunits. Instead of a high affinity binding site for benzodiazepines, a second interface of interest exists, as well as an interface formed by a (+) and a (-) side.
Classical benzodiazepines, like diazepam, predominantly interact with receptors composed of 12, 22, 32 or 52. They show no activity on 42 or 62 receptors and show a reduced activity on receptors containing 1 or 3 subunits (Sieghart 1995), (Khom, Baburin et al. 2006). In in-vivo studies with genetically modified mice, where single subunit types were mutated thus forming receptors insensitive to diazepam, it has been shown that diazepam has specific pharmacological effects depending on which subunit forms the benzodiazepine binding site (Rudolph and Mohler 2004); (Atack 2005); (Whiting 2006). Consequently it has been shown that via 12 receptors diazepam mediates sedative and anticonvulsive effects (Rudolph, Crestani et al. 1999), via 22 or 32 receptors it mediates anxiolytic effects (Low, Crestani et al. 2000); (Dias, Sheppard et al. 2005) and via 52 receptors learning and memory effects (Rudolph and Mohler 2004).
Ligands mediating their effects via the benzodiazepine binding site are not able to directly activate GABAA receptors. Therefore these drugs exhibit low toxicity.
16
Introduction
Unfortunately, chronic use of benzodiazepines in the course of epilepsy leads to tolerance development – benzodiazepines thus become in-effective in mediating anticonvulsive effects (Sieghart 1995). While the mechanisms leading to tolerance are not well understood, one possible mechanism could be the downregulation of receptors and replacement by receptors.
In addition to benzodiazepines, many other compounds belonging to different compound classes are able to bind at the GABAA receptor and allosterically modulate GABA-induced currents. For most of these compounds no binding site has been unequivocally identified. Additional binding sites located at the +- or
+- interfaces of the extracellular domain of the GABAA receptor are very likely, but have not been investigated so far(Ramerstorfer, Furtmuller et al. 2011).
3.4 Aim of this thesis
In this thesis we investigated the +- interface as a possible novel drug- binding site of the GABAA receptor. Because the +- interface consists of the same + side as the +- interface, which builds the benzodiazepine binding site, we speculated that at least some ligands of the benzodiazepine binding site might also bind at the +- interface. To avoid interaction with the benzodiazepine binding site, we investigated whether benzodiazepine binding site ligands are active at 13 receptors that do not contain a high affinity benzodiazepine binding site. Ligands identified should then be investigated for their possible interaction with the +- interface by a steric hindrance approach. After introducing cysteines at several interface-lining positions of the plus side of the 1 and the minus side of the 3 subunit (Fig. 4) their labeling by a cysteine reactive reagent named MTSEA-biotin should reduce or completely block the binding of drugs at a possible binding site at the +- interface.
Compounds acting as positive allosteric modulators at GABAA receptors via the
+- interface would modulate all sub-populations of GABAA receptors and could be used as anticonvulsants. Since a subunit is not required for their action they possibly might avoid tolerance development. In addition, compounds selectively interacting with interfaces containing specific and subunits might
17
Introduction exhibit novel and quite specific actions, and might become as important as the benzodiazepine site ligands.
18
Materials and Methods
4 MATERIALS AND METHODS
4.1 Chemicals
Compounds were obtained from the following sources: diazepam (7- chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) Sigma; CGS 9895 (2-p-methoxyphenylpyrazolo[4,3-c]quinolin-3(5H)-one) was a gift of Ciba Geigy; Summit, NJ, USA; PZII-028 was a gift of Jim Cook, University of Milwaukee, Wisconsin, USA; MTSEA-biotin (N-biotinoylaminoethyl methanethiosulfonate) was purchased from Toronto Research Chemicals, Ontario, Canada. All compounds used in the screening (Fig. 5) were gifts of James M. Cook.
4.2 GABAA receptor subunits and point mutations
cDNA´s of rat GABAA receptor subunits 1, 3, and 2S were cloned as described (Ebert, Scholze et al. 1996). cDNAs of the rat subunits 2, 3, and 5 were gifts from P. Malherbe. The mutated constructs 1F99C, 1H101C, 3N41C, 3D43C, 3A45C, 3Y62C, 3Q64C and 2M130C were generated as described (Tan, Gonthier et al. 2007) by Isabella Sarto-Jackson and the mutated constructs 1S204C, 1S205C, 1T206C, 1V211C, 1S290N and 2F77I were gifts from Erwin Sigel. For the generation of mutated 1, 3 and 2 subunits, these subunits were subcloned into pCI expression vectors (Promega, Madison, WI, USA) as described previously (Tretter, Ehya et al. 1997). Mutated subunits were constructed by PCR amplification using the wild-type subunit as a template. For this, PCR primers were used to construct point mutations within the subunits by the ‘gene splicing by overlap extension’ technique (Horton, Ho et al. 1993). The PCR primers for 1F99C, 1H101C, 3N41C, 3D43C, 3A45C, 3Y62C, 3Q64C and 2M130C contained XmaI and XhoI restriction sites, which were used to clone the 1, 3 or 2 into pCI vector, respectively
19
Materials and Methods
(Promega, Madision, WI, USA). The mutated subunits were confirmed by sequencing.
4.3 Two electrode voltage clamp:
For heterologous expression of GABAA receptors, oocytes of Xenopus leavis were used. By injecting either DNA into the nucleus or mRNA into the cell, oocytes are able to express the respective protein. Since the intra-nucleus injection of DNA has a success-rate of approximate 10 to 15%, whereas mRNA injection gives a success-rate of almost 100% we favoured the mRNA injection. For that we had to synthesize capped RNA (containing a 7-methyl guanosine cap structure at the 5’ end) mimicking the real mRNA, by using in vitro transcription of the respective DNA produced for the different constructs.
4.3.1 In vitro transcription of capped cRNA In vitro transcription of mRNA was based on the cDNA expression vectors encoding for wild-type GABAA receptor subunits 1, 2, 3, 5, 1, 2, 3 and 2 as well as for mutated subunits 1F99C, 1H101C,1S204C, 1S205C, 1T206C, 1V211C, 1S290N, 3N41C, 3D43C, 3A45C, 3Y62C, 3Q64C and 2M130C (all from rat).
4.3.1.1 Linearizing cDNA vectors 10µg of the respective cDNA vector was linearized with the restriction endonucleases NotI for 1, 2, 3, 5, 1, 2, 3, 1F99C, 1H101C, 3N41C, 3D43C, 3A45C, 3Y62C and 3Q64C, HindIII for 1S204C, 1S205C, 1T206C, 1V211C, S290N and BamHI for 2. The complete digestion mix contained the respective cDNA and 10-times buffer depending on the restriction enzyme (buffer H for NotI, and buffer E for HindIII (Promega,WI) and buffer B for BamHI (Roche, CH) and H2O to a total volume of 100µl. cDNA was digested over night (o/n) at 37°C.
20
Materials and Methods
4.3.1.2 Proteinase K-digestion To remove remaining restriction endonucleases, 0,7µl of Proteinase K (Ambion, Austin, TX) was added to the restriction mix and incubated for 30 min at 37°C.
4.3.1.3 Extraction of cDNA To remove salts and remaining enzymes of the extraction mix it is necessary to extract and precipitate the construct. All steps of extraction were done at room temperature (RT). To the digested cDNA we added the same volume (100µl) of phenol / chloroform (Sigma Aldrich, MO) at a ratio of 1:1 for phenol and chloroform at a pH of 8,0. The mixture was vortexed for at least 30 sec and centrifuged for 5 min at approximately 15.700xG (at maximum speed of an Eppendorf centrifuge). The DNA is in the upper, aqueous phase, and can be removed by a pipette and transferred to a new vial. This had to be repeated. Finally, the cDNA was extracted with 100µl pure chloroform, vortexed, and centrifuged as before.
4.3.1.4 Precipitation of cDNA by ethanol To the remaining extract 10µl of NaAc (3M sodium acetate at pH 5,5; Ambion), and 250µl of 100%-pure ethanol (EtOH) was added. Mixture was vortexed and placed to -80°C for 10 min and at -20°C for 30min. Vial was centrifuged for 30min at 4°C at maximum rpm. The supernatant was removed and the extract was carefully washed twice with EtOH at 75%. The pellet was then dried to remove the remaining EtOH. The pellet was then dissolved in 12µl of RNAse free Tris-EDTA (TE) buffer (Sigma Aldrich, MO). Concentration estimation was done by using a Picogreen (Invitrogen, CA) assay.
4.3.1.5 Transcription By using the Message Machine T7 kit (Ambion, TX) capped cRNA transcripts of the respective constructs were synthesized. All buffers and reagents were used from the Message Machine kit. The transcription mix contained 1µg of linearized DNA, 2µl of 10-times buffer, 10µl of 2-times nucleotide triphosphate –CAP (NTP/CAP), 2µl of the enzyme mix and RNAse free H2O to complete a total volume of 20µl. This mix was then incubated for 2 h
21
Materials and Methods at 37°C. To end the transcription we added 2µl of DNAse I (Kit) to the transcription mix and incubated for 15min at 37°C.
4.3.1.6 Extraction and Precipitation of RNA To increase the volume of the RNA mix to a reasonable size we added 95µl of TE. Then15µl (10% of the total volume) of Ammonium Acetate Stop Solution was added to the mix. The steps of the extraction are the same as before at the DNA extraction, but here we used phenol chloroform at a pH of 4.2, because of the RNA instead of the DNA.
After the last step with chloroform, we added here 150µl of isopropanol to aqueous phase, heavily mixed it and put it to -20°C for 20min. Then we removed the isopropanol and washed the pellet with EtOH at 75%. After that we dried the pellet to remove the EtOH and dissolved it in 30µl of TE.
4.3.1.7 Polyadenylation To improve the stability of the cRNA transcripts we added a poly (A) tail by using yeast poly (A) polymerase (USB, OH). Here we added all 30µl of RNA in TE to 12µl 5-times buffer (USB, OH), 4µl of ATP (Amersham, UK) at 10mM and 1.5µl of Poly (A) polymerase and incubated this mix for 15min at 30°C. We added 120µl of TE to increase the total volume to a reasonable scale.
4.3.1.8 Extraction and precipitation of mRNA The very same steps as in 4.2.1.6 were repeated here. But after the last chloroform extraction the aqueous phase containing the mRNA was transferred in Non-stick RNAse free vials (Ambion). Then 12µl of NaAC (3M, pH 5,5) and 400µl of 100% EtOH were added and mRNA was precipitated over night at - 20°C. Precipitate was centrifuged and washed with 75% EtOH at 4°C. Pellet was dried and afterwards dissolved in 20µl diethylpyrocarbonate-treated water
(DEPC-H2O) (Ambion). A Ribogreen (Invitrogen, CA) assay was performed for quantification. The integrity of the transcript was determined by electrophoresis with a 0,9% agarose/formaldehyde gel by staining with Radiant Red RNA Stain (Bio Rad) by comparing with a molecular weight marker (RNA Ladder; Gibco
BRL) (Sigel 1987). mRNA was diluted and stored in DEPC-H2O at -70°C.
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Materials and Methods
4.3.2 Protein expression in Xenopus leavis oocytes The methods for isolating, culturing, injecting and defolliculating of oocytes were identical with those described by E. Sigel (Sigel, Baur et al. 1990) and were described elsewhere (Li, Cao et al. 2003). Mature female Xenopus laevis (Nasco, WI) were anaesthetized in a bath of ice-cold 0.17 % Tricain (Ethyl-m-aminobenzoat, Sigma Aldrich, MO) before decapitation and removal of the frog’s ovary. Lobes of ovaries were isolated from the frog. Within the lobes oocytes are connected by blood vessels in a connective tissue. For the use in electrophysiology it is necessary to single out the cells at the mature stage 5 to 6. At this stage the oocyte appears with two different hemispheres, a dark brown (animal) and a yellowish (vegetal) hemisphere. The animal hemisphere contains the nucleus whereas the vegetal hemisphere contains predominantly yolk. Lobes of ovary were positioned in a petri dish in Xenopus Ringer solution
(XR, containing 90 mM NaCl, 5 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1mM
KCl and 1 mM CaCl2) under a stereo microscope. With the follicle cell layer around them, oocytes were singled out using a platinum wire loop. Oocytes were stored and incubated at 18°C in modified Barths’ Medium (88mM NaCl,
10mM HEPES-NaOH (pH 7.4), 2.4 mM NaHCO3, 1mM KCl, 0.82 mM MgSO4,
0.41 mM CaCl2, 0.34 mM Ca(NO3)2) that was supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. To avoid damage of the nucleus, aqueous solution of mRNA was injected into the vegetal hemisphere of the oocytes with follicle cell layer still around them. A total of 2.5ng of mRNA per oocyte was injected. Subunit ratio was 1:1:5 for all receptors and 1:1 for all receptors consisting of the different variations of wild-type and mutated subunit combination used in this study. After injection of mRNA, oocytes were incubated for at least 24 hours for 13 receptors and for at least 36 hours for 132 receptors before the enveloping follicle cell layers were removed. Collagenase-treatment (type IA, Sigma Aldrich, MO) and mechanically defolliculating of the oocytes was performed as described in (Li, Cao et al. 2003).
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Materials and Methods
4.3.3 Electrophysiological recordings For electrophysiological recordings, oocytes were placed on a nylon-grid in a bath of XR. The oocytes were constantly washed by a flow of 6 ml/min XR which could be switched to XR containing GABA and/or drugs. Drugs were diluted into XR from DMSO-solutions resulting in a final concentration of 0.1 % DMSO perfusing the oocytes. Drugs were pre-applied for 30 sec before the addition of GABA, which was then co-applied with the drugs until a peak response was observed. Between two applications, oocytes were washed in XR for up to 15 min to ensure full recovery from desensitization. For current measurements the oocytes were impaled with two microelectrodes (2-3 M) which were filled with 2 M KCl. Maximum currents measured in mRNA injected oocytes were in the microampere range for all subtypes of GABAA receptors. To test for modulation of GABA induced currents by drugs, a concentration of GABA that was titrated to trigger 3% of the respective maximum GABA-elicited current of the individual oocyte (EC3) was applied to the cell with various concentrations of drugs. 2mM MTSEA-biotin (N-Biotinylaminoethyl Methanethiosulfonate) solution was freshly made in XR containing the respective GABA-EC3 concentration. Cells were immediately pre-incubated in MTSEA-biotin solution for 3 min and washed with XR for 5 min. All recordings were performed at room temperature at a holding potential of -60mV using a Warner OC-725C two-electrode voltage clamp (Warner Instrument, Hamden, CT) or a Dagan CA-1B Oocyte Clamp or a Dagan TEV-200A two-electrode voltage clamp (Dagan Corporation, Minneapolis, MN). Data were digitized, recorded and measured using a Digidata 1322A data acquisition system (Axon Instruments, Union City, CA). Data were analyzed using GraphPad Prism (GraphPad Software, CA, USA). Data for GABA dependent dose response curve were fitted to the equation Y=Bottom+(Top-Bottom)/1+10(LogEC50-X)*nH, where EC50 is the concentration of the compound that increases the amplitude of the GABA-evoked current by 50%, and nH is the Hill coefficient. Data are given as mean ± S.E. from at least three oocytes and 2 oocyte batches. Statistical significance was calculated using unpaired Student’s t test or a one- way ANOVA with a Bonferroni’s post test.
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Results
5 RESULTS
Parts of the text, figures and legends have been taken from (Ramerstorfer, Furtmuller et al. 2011)
5.1 The pyrazoloquinoline CGS 9895 enhances GABA- induced chloride flux in 13 receptors
Both, the +- interface and the benzodiazepine binding site at the +- interface contain the + side (Fig. 4A). A binding site at the +- interface might thus also accommodate at least some of the benzodiazepine site ligands. To identify drugs possibly mediating some of their effects via the +- interface and to avoid interaction with the benzodiazepine binding site, we therefore used
GABAA receptors composed of 1 and 3 subunits, only. Such receptors are assumed to be composed of 3 and 2 subunits (Tretter, Ehya et al. 1997; Farrar, Whiting et al. 1999; Baumann, Baur et al. 2001), and should thus have 2 3+/1- interfaces (GABA binding sites), 2 1+/3- interfaces, and one 3+/3- interface, but no benzodiazepine binding site (Fig. 4B introduction). In a screening of 95 newly synthesized compounds belonging to different substance classes such as benzodiazepines, imidazo-benzodiazepines and pyrazoloquinolines (Fig. 5) at a concentration of 1 M at GABAA receptors consisting of 1 and 3 subunits, the pyrazoloquinoline PZ-II-028 was able to strongly potentiate GABA-induced currents. After a search for related compounds we found several substances all synthesized by the company Ciba Geigy, which now belongs to Novartis. We as well screened these related substances in the same manner as the 95 compounds before, and out of these substances the anxiolytic pyrazoloquinoline CGS 9895 (Fig. 6A) (Bennett 1987) was able to show the strongest enhancement of GABA-induced currents in recombinant 13 receptors expressed in Xenopus oocytes.
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Results
Fig. 5 Screening of possible allosteric modulators at binding sites independent of the BZ-binding site. 95 compounds at 1µM were co-applied with a GABA concentration at EC3 at 13 receptors. Red dashed line represents threshold of 100% that equals the GABA EC3 effect alone. Positive allosteric modulators have stimulations above the threshold, negative modulators below the threshold and possible non-binders as well as allosteric antagonists will display effects of around 100%. Effect of the pyrazoloquinoline PZII-028 (red bar) showing a 370% modulation of EC3 GABA current.
Due to the fact that the pyrazoloquinoline CGS 9895 was a well known benzodiazepine site ligand with anxiolytic properties we focused our
26
Results investigation at this compound and set back PZII-028 belonging to the same substance class to investigate it in future studies.
Fig. 6 Structure and pharmacology of CGS 9895. (A) Structure of CGS 9895. (B) Concentration dependent effect of CGS 9895 on 13 and 132 receptors at GABA EC3 and EC20. 30 µM of CGS 9895 stimulated GABA evoked currents (GABA EC3) in 13 and 132 receptors to 615±61% (n=5) and 660±49% (n=5). This is not significantly different. At GABA EC20, 30µM CGS 9895 enhanced currents in 13 (n=4) and 132 (n=6) to 262±5% and
349±22%. This is significantly different (p<0.05). CGS 9895 stimulation of 13 and 132 receptors at GABA EC3 or
EC20 was significantly different at 3 µM (n=4, p<0.05), 10 µM (n=4-10, p<0.001), and 30 M (n=4-6, p<0.01).
Subsequent concentration-response curves indicated that CGS 9895 started to enhance GABA-induced currents (GABA EC3) in these receptors at 1 M. At 10 M, this compound was able to stimulate this current up to 400%, and at 30 M up to 600% (Fig. 6B). CGS 9895, however, did not directly elicit a chloride current in the absence of GABA.
5.2 CGS 9895 is an antagonist at the benzodiazepine binding site
Since this compound in radioligand displacement studies has been demonstrated previously to exhibit a low nM affinity for the benzodiazepine binding site of GABAA receptors (Yokoyama, Ritter et al. 1982; Brown and
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Results
Martin 1984), we compared its action at 13 and 132 receptors. CGS 9895 did not modulate GABA-induced currents at nM concentrations in 132 receptors, but at M concentrations elicited a comparable current enhancement in 13 and 132 receptors (Fig. 6B). This effect could be observed at GABA concentrations eliciting 3% (GABA EC3) or 20% (GABA EC20) of the maximal GABA current in the respective oocytes (Fig. 6B). In addition, 10 M CGS 9895 elicited a left-shift in the GABA concentration-response curve in 13 and 132 receptors (Fig. 7A and B).
Fig. 7 GABA concentration-response curves in the absence (■) or presence (□) of 10µM CGS 9895. Effects are normalized to the maximum evoked GABA current. 10µM CGS 9895 evokes a left-shift of the GABA EC50 value from 11 to 4 µM (p<0.001) at 13 receptors (A) and from 73 to 33µM (p<0.001) at 132 receptors (B). The experiments were performed 6-8 times in different oocytes.
Since the CGS 9895 effect was much stronger at GABA EC3 than at GABA
EC20, all future experiments were performed at GABA EC3. CGS 9895 thus seems not to need the benzodiazepine binding site for producing its effect on these GABAA receptors. This conclusion was confirmed by experiments indicating that the effect of 10 M CGS 9895 on 132 receptors in contrast to that of 10 M diazepam could not be inhibited by a 10 M concentration of the benzodiazepine site antagonist Ro15-1788 (Fig. 8A). It was thus possible that
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CGS 9895 acts as antagonist at the benzodiazepine binding site and stimulates these receptors via a second, so far unknown site, which is also present at 13 receptors. This conclusion was confirmed by experiments demonstrating that 50 nM CGS 9895, a concentration that completely saturates the benzodiazepine binding site of GABAA receptors but does not stimulate GABA- induced currents, was able to completely inhibit the effects of diazepam on 132 receptors (Fig. 8B). These data are in agreement with previous results indicating that CGS 9895 is an antagonist at the benzodiazepine binding site (Brown and Martin 1984; Bennett 1987).
Fig. 8 CGS 9895 is an antagonist at the benzodiazepine site and enhances GABA-induced currents via a different site at 132 receptors. (A) Modulation of GABA EC3 currents of recombinant 132 receptors by 10 M CGS 9895 (upper graph) or 10 M diazepam (lower graph) in the absence or presence of 10 M Ro 15-1788. The experiments were performed ten times with comparable results (B) Concentration-response curves for diazepam (X), CGS 9895 (■), and diazepam together with 50nM CGS 9895 (□) on 132 receptors. CGS 9895 at 50nM does not stimulate GABA evoked currents (○). The experiment was performed four times with comparable results.
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5.3 A steric hindrance approach indicates that CGS 9895 does not mediate its action via the +- interface
To finally exclude the possibility that CGS 9895 exerts its potentiating action via the +- site in 132 receptors, the interaction of this drug with the benzodiazepine binding site was inhibited by using the substituted cysteine accessibility method for introducing a steric hindrance into the pocket. For that, several amino acid residues at the 2- side of the pocket were selected by using our homology model of GABAA receptors as a guide (Ernst et al., 2003) and individually mutated to cysteines. The effects of these mutations on GABA- induced current and its modulation by diazepam were then investigated in Xenopus oocytes expressing these mutated receptors. Introduction of cysteines into binding pockets in many cases impairs the function of receptors (see 5.4.), making interpretation of subsequent steric hindrance experiments difficult. Therefore, we selected those point mutations that produced no or small changes in the properties of the receptors for our experiments.
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Fig. 9 The effects of a steric hindrance introduced into 132 receptors via 2M130C on GABA, diazepam, and CGS 9895 concentration-response curves. Symbols used for 132 receptors (□), 132 receptors plus MTSEA-biotin (◊), 132M130C receptors (■) and 132M130C receptors labelled with MTSEA-biotin (X). (A) GABA effects normalized to the maximum evoked GABA current. (B) GABA evoked currents. Due to the variability of the data (expression levels of receptors) the differences observed in the different concentration-response curves are not significant (n=3-8) (C) Diazepam effects on 132 and 132M130C receptors in the absence or presence of MTSEA-biotin. Upper graph: concentration-response curves of diazepam (n= 3-5). MTSEA-biotin caused no significant difference in the diazepam effects on 132 receptors, but a significant difference in the diazepam effects on
132M130C receptors at 100 nM, 1M, or 10 M ( p<0.05). Lower graph: individual current traces at GABA EC3 in the absence or presence of 10 M diazepam under the conditions indicated. (D) Effects of CGS 9895 on 132 and 132M130C receptors in the absence or presence of MTSEA-biotin. Upper graph: concentration-response curves of
CGS 9895 (n=3-8). Lower graph: individual current traces at GABA EC3 in the absence or presence of 10 M CGS 9895 under the conditions indicated.
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The point mutation 2M130C, although causing an approximately 2fold reduction in GABA potency (Fig. 9A, Table 1), did not significantly change the potency and efficacy of diazepam (Fig. 9C) or CGS 9895 (Fig. 9D) to stimulate the mutated receptor. The apparent reduction in GABA-induced currents indicated in Fig. 9B was not significant due to the variability of data possibly caused by different expression levels of receptors in different oocytes. Wild-type and mutated receptors were then exposed to MTSEA-biotin (2 mM) for 2 min, and GABA-elicited currents as well as their potentiation by diazepam or CGS 9895 were measured before and after MTSEA-biotin exposure (Teissere and Czajkowski 2001). In wild-type 132 receptors MTSEA-biotin had no significant effects on GABA (Figs. 9A, B), diazepam (Fig. 9C), or CGS 9895 (Fig. 9D). Thus, any effect of this reagent on mutated receptors must have been due to covalent labelling of the introduced cysteines. After MTSEA-biotin treatment the GABA dose-response curve of the mutated receptor was shifted to the left and the potency of GABA for opening the chloride channel was increased (Figs. 15A, B; Table 1), suggesting covalent labelling of the introduced cysteine and possibly indicating that MTSEA-biotin incorporated into the benzodiazepine site at least partially can cause conformational changes comparable to that of benzodiazepines (Teissere and Czajkowski 2001).
MTSEA-biotin nearly eliminated the stimulation of GABA EC3 by diazepam in the mutated receptor (Fig. 9C), but did not influence the stimulation of this receptor by CGS 9895 (Fig. 9D). Together, these results indicated that CGS 9895, in contrast to diazepam, did not produce its GABA-agonistic effect via the benzodiazepine binding site. In addition, these results demonstrate that the action of a drug at a binding site can be efficiently inhibited by such a steric hindrance approach without influencing the effects of drugs interacting with another binding site.
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5.4 Identification of suitable mutations for steric hindrance at the +- interface
To investigate whether CGS 9895 is mediating its potentiating action at
GABAA receptors via the +- interface, we applied a similar steric hindrance approach to this interface. For that, several pocket forming amino acid residues located at the 1+ or the 3- side were mutated to cysteines.
Fig. 10 Outside view onto the +- interface of the extracellular domain of the GABAA receptor (Sieghart, Ramerstorfer et al. 2011). Selected interface spanning amino acid residues at the + side (yellow) and - side (red) are highlighted. At + side, residues F99 and H101 at loop A, residues S204, S205, T206 and V211 at loop C. At the - side, residues A45 and Q64C at loop D. Residues used in steric hindrance experiments are shown in yellow labels, and those affecting the ligand potency or efficacies upon mutagenesis are shown in grey labels.
13 or 132 receptors containing the respective mutated subunits were expressed in Xenopus oocytes and investigated for the effect of the mutation on GABA-induced currents and their stimulation by diazepam or CGS 9895. As mentioned above, introduction of cysteines into binding pockets in many cases impairs the function of receptors, making interpretation of subsequent steric hindrance experiments difficult. For our studies we therefore selected those
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Results point mutations that produced no, or only small changes in the properties of the receptors.
Fig. 11 Steric hindrance introduced via 1S204C inhibits the effect of CGS 9895. Symbols used for 13 (○),1S204C3 (■) and 1S204C3 receptors labelled by MTSEA-biotin (X). (A) GABA effects normalized to the maximum evoked currents. (B) GABA evoked currents. (C) Concentration-response curves of CGS 9895 on 13,1S204C3 and 1S204C3 receptors labelled by MTSEA-biotin . The experiments were performed 3- 5 times in different oocytes. The reduction of the CGS 9895 effect by MTSEA-biotin in 1S204C3 receptors was statistically significant at 10 M (p<0.001).
The mutation 1S204C did not change the potency of GABA (Fig. 11A, B). It showed a slight but not statistically significant reduction of CGS 9895 effect at 1S204C3 receptors (Fig. 11C) but modification with MTSEA-biotin significantly reduced the CGS 9895 effect at the mutated receptor by 51%. The
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Results mutation 1S204C would thus be a good candidate for the steric hindrance approach.
Fig. 12 Steric hindrance at 1V211C reduces CGS effect at receptors but not at receptors. (A) GABA effects normalized to the maximum evoked currents at receptors. (B) GABA evoked current at receptors. (C) Concentration-response curves of CGS 9895 on 13 (○), 1V211C3 (■) and 1V211C3 receptors labelled by MTSEA-biotin (X). Each experiment was performed 4 times in different oocytes. The effect of CGS 9895 was significantly reduced by the MTSEA-biotin at 10 M (P<0.001).
The mutation 1V211C did not significantly change the potency of GABA (Fig. 12A) but enhanced the GABA-induced current in 1V211C3 receptors. This could either have been due to an increase of the expression or of the conductance of receptors (Fig. 12B). In any case, this enhancement of the
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GABA current in the mutated receptor was inhibited in the presence of MTSEA- biotin. Interestingly, the mutation 1V211C3 did not change the effect of CGS 9895 as compared to wild-type receptors and could thus be used for investigating a possible steric hindrance at the CGS 9895 binding site. As indicated in Fig. 12C, MTSEA-biotin was able to reduce the effect of CGS 9895 by 60% in these receptors.
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Fig. 13 Steric hindrance at 3Q64C strongly reduces CGS 9895 effect. GABA effects (n=3-4) normalized to the maximum evoked currents at (A) receptors and at (B) receptors. GABA evoked current at (C) receptors and at (D) receptors. (E) Concentration-response curves of CGS 9895 on 13 (○), 13Q64C (■) and 13Q64C receptors labelled by MTSEA-biotin (X). Each experiment was performed 5-11 times in different oocytes. The effect of CGS 9895 was significantly reduced by MTSEA-biotin at 10 M (P<0.01). (F) Concentration-response curves of CGS 9895 on 132 (□), 13Q64C2 (■) and 13Q64C2 receptors labelled by MTSEA-biotin (X). Each experiment was performed 3-10 times in different oocytes. The effect of CGS 9895 was significantly reduced the point mutation (P<0.05) and MTSEA-biotin reduced the CGS 9895 effect even further (P<0.001).
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Results
The mutation 3Q64C did not significantly change the potency of GABA (Fig. 13A, B) in and receptors, but there was some change in the GABA- induced currents in these receptors. However, this mutation did not influence the stimulation of the GABA current by CGS 9895 in receptors and the modification by MTSEA-biotin shows a strong 50% reduction of compound effect. At receptors (Fig. 13F) the mutation shows already a significant reduction of CGS 9895 effect, and MTSEA-biotin showed a further reduced compound effect. Due to these data and the fact that MTSEA-biotin could inhibit CGS 9895 effects in both 13Q64C and 13Q64C2 receptors we decided to use the 3Q64C mutation for our steric hindrance experiments at the 3- side.
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5.5 Application of the steric hindrance approach using two suitable point mutations at the +- interface
To investigate the effects of the mutation 3Q64C on the modulation of
GABAA receptors by diazepam and CGS 9895 we expressed 13Q64C2 receptors.
Fig. 14 Effects of a steric hindrance introduced into 132 receptors via 3Q64C on diazepam and CGS 9895 concentration-response curves. Symbols used: for 132 receptors (□), 132 receptors plus MTSEA-biotin (◊), 13Q64C2 receptors (■), and 13Q64C2 receptors labelled with MTSEA-biotin (X). (A) Diazepam effects on 132 and 13Q64C2 receptors, the latter in the absence or presence of MTSEA-biotin. Upper graph: concentration-response curves of diazepam (n= 4-5). MTSEA-biotin caused no significant difference in the diazepam effects. Lower graph: individual current traces at GABA EC3 in the absence or presence of 10 M diazepam under the conditions indicated. (B) Effects of CGS 9895 on 132 and 13Q64C2 receptors, the latter in the absence or presence of MTSEA-biotin. Upper graph: concentration-response curves of CGS 9895 (n=3-10). There was a significant reduction of the effects of 10 M CGS 9895 in the point mutated receptor (p<0.05) and this reduction was even enhanced in the presence of MTSEA-biotin (p<0.001). Lower graph: individual current traces at GABA EC3 in the absence or presence of 10 M CGS 9895 under the conditions indicated.
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Whereas this mutation did also not significantly reduce the potency and efficacy of diazepam for stimulation of these receptors (Fig. 14A), the effects of CGS 9895 were significantly reduced by the point mutation in 13Q64C2 receptors (Fig. 14B). On incubation with MTSEA-biotin, GABA-induced currents (Figs. 14A, B) as well as their stimulation by diazepam (Fig. 14A) were not significantly changed but the effect of CGS 9895 was further reduced (Fig. 14B), again suggesting that CGS 9895 might exert its action via the +- interface.
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Table 1. GABA EC50 and Hill slopes (nH) of different GABAA receptors in the absence or presence of 2mM MTSEA- biotin
Receptor GABA EC50 (µM) Hill slope
13 11±1.3 0.85
13 MTSEA-biotin 12±2.2 0.76
13 + 10µM CGS 9895 4.2±0.3 0.81
1V211C3 4.9±0.5 0.81
1V211C3 MTSEA-biotin 12.8±1.9 0.72
13Q64C 17.1±7.3 0.55
13Q64C MTSEA-biotin 8.6±0.8 0.70
1V211C3Q64C 19.6±1.5 0.65
1V211C3Q64C MTSEA-biotin 23.8±5.9 0.61
132 73.4±4.2 1.17
132 MTSEA-biotin 59.1±22.1 1.08
132 +10µM CGS 9895 32.5±3.8 1.05
13Q64C2 81.9±11 0.94
13Q64C2 MTSEA-biotin 98.5±39.5 0.84
132M130C 225.1±41.6 0.84
132M130C MTSEA-biotin 99.4±49.2 0.99
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If this is the case, it should be possible to block the action of CGS 9895 also via the 1 subunit. To avoid an interaction of CGS 9895 with both the +- interface and the benzodiazepine binding site located at the +- interface, we therefore repeated the steric hindrance approach with GABAA receptors composed of 1 and 3 subunits, only. For that we expressed either 13Q64C or 1V211C3 receptors and investigated the effect of covalent modification of these mutants by MTESA-biotin. As shown in Fig. 15A, the GABA-stimulatory effect of CGS 9895 in 13Q64C was not different from that in 13 receptors, but covalent modification of the introduced cysteine by MTSEA-biotin was able to reduce this effect by 50%. In contrast, MTSEA-biotin did not significantly change the effects of CGS 9895 in wild-type 13 receptors (Fig. 15A). Similarly, the mutation 1V211C did not significantly change the potency of GABA and did not change the effects of CGS 9895 on GABA-induced currents (Fig. 15B). On covalently labelling the introduced cysteine by MTSEA-biotin, however, the CGS 9895-induced enhancement of GABA currents was reduced by 50% (Fig. 15B).
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Fig. 15 Steric hindrance introduced via 3Q64C or via 1V211C inhibits the effect of CGS 9895. (A) Concentration-response curves of CGS 9895 on 13 (○), 13 receptors plus MTSEA-biotin (◊), 13Q64C (■) and 13Q64C receptors labelled by MTSEA-biotin (X). Each experiment was performed 5-11 times in different oocytes. The reduction of the CGS 9895 effect by MTSEA-biotin in 13Q64C receptors was significant at 10 M (p<0.01) (B) Concentration-response curves of CGS 9895 on 13 (○), 13 receptors plus MTSEA-biotin (◊), 1V211C3 (■) and 1V211C3 receptors labelled by MTSEA-biotin (X). The experiments were performed 5-11 times in different oocytes. The reduction of the CGS 9895 effect by MTSEA-biotin in 1V211C3 receptors was significant at 10 M (p<0.001)
In other experiments it was demonstrated that modification of position 1S204C by MTSEA-biotin also reduced CGS 9895 stimulation (Fig. 11C). Finally, we investigated the effect of CGS 9895 in 1V211C3Q64C receptors, in which cysteines were introduced at the 1+ as well as at the 3- side. As shown in Fig. 16A, the two introduced cysteines did not significantly influence the GABA- stimulatory effect of CGS 9895, but covalent labelling of cysteines with MTSEA- biotin nearly completely abolished this effect. In contrast, the effects of ROD 188 (Thomet, Baur et al. 2000), interacting with a so far unknown binding site at the GABAA receptors (Fig. 16B), on 13 receptors were neither changed by the introduced cysteines at the 1+ or 3- side, nor by their covalent modification by MTSEA-biotin, suggesting that receptors were not non- specifically inactivated by MTSEA-biotin. Together, these data strongly suggest that CGS 9895 exerts its action via the extracellular part of the 1+3- interface.
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Fig. 16 Steric hindrance introduced via both mutations inhibits the effect of CGS 9895 but not that of ROD 188 on 13 receptors. (A) Upper graph: Concentration-response curves of CGS 9895 on 13 (○), 13 receptors plus MTSEA-biotin (◊), 1V211C3Q64C (■) and 1V211C3Q64C receptors labelled by MTSEA-biotin (X) The experiments were performed 5-10 times in different oocytes. The reduction of the CGS 9895 effect by MTSEA-biotin in
1V211C3Q64C receptors was significant at 10 M (p<0.05). Lower graph: individual current traces at GABA EC3 in the absence or presence of 10 M CGS 9895 under the conditions indicated. (B) Upper graph: Concentration-response curves of ROD 188 on 13 (○), 13Q64C (▲), 1V211C3 (■), 1V211C3Q64C (▼), MTSEA-biotin labelled
13Q64C (∆), MTSEA-biotin labelled 1V211C3 (□), and MTSEA-biotin labelled 1V211C3Q64C (∇) receptors. The experiments were performed 3-5 times in different oocytes, and there were no significant differences between the curves (wild type vs. mutated receptors, or mutated receptors vs. mutated receptors in the presence of MTSEA biotin).
Lower graph: individual current traces at GABA EC3 in the absence or presence of 10 M ROD 188 under the conditions indicated.
In an opposite approach we considered the possibility to inhibit covalent labelling of the introduced cysteines by MTSEA-biotin in the presence of CGS 9895. However due to the irreversible nature of the covalent modification by MTSEA-biotin that within a very short time overrules the reversible binding of CGS 9895, and due to simultaneous and partially opposite changes in currents induced by GABA and CGS 9895 during this reaction, such experiments cannot be correctly interpreted.
44
Results
To investigate whether the effect of CGS 9895 was mediated via the 3+3- interface that also is present in 13 receptors, Xenopus oocytes were injected with 3 subunits, only, and the resulting homo-oligomeric receptors were investigated. In agreement with previous results, the ion channel formed by these homo-oligomeric 3 receptors were open in the absence of GABA, and could be modulated by pentobarbital and be blocked by picrotoxin (Slany, Zezula et al. 1995; Wooltorton, Moss et al. 1997). In contrast, CGS 9895 was not able to change the currents mediated by this channel (experiments not shown), indicating that either no binding site for this compound can be formed by the 3+3- interface in these receptors or that CGS 9895 modulation of this channel via this interface is not possible.
In previous studies (Walters, Hadley et al. 2000) it was demonstrated that diazepam at concentrations >10 M was able to potentiate GABA-induced currents in 12 receptors. We demonstrated a similar potentiation of GABA- induced currents by 30-100 M diazepam on 13 receptors. The effects at 100 M diazepam were similar in 13 and 1V211C3Q64C receptors, but could not be significantly inhibited by MTSEA-biotin (Fig. 17) in contrast to the CGS 9895 effects. This is consistent with the suggestion that this low potency diazepam effect possibly might be caused by an interaction with the transmembrane domain of the receptor (Walters, Hadley et al. 2000; Baur, Tan et al. 2008).
45
Results
Fig. 17 Diazepam effect at 13 receptors is neither affected by mutations at 1 or 3 subunits nor by steric hindrance at these mutants. Concentration-response curves of diazepam on 13 (○), 1V211C3Q64C (■) and 1V211C3Q64C receptors labelled by MTSEA-biotin (X). The experiments were performed 6-8 times in different oocytes. The reduction of the diazepam effect by mutations or MTSEA-biotin in 1V211C3Q64C receptors was not significant at 100 M.
While this work was in progress, evidence was presented indicating that flurazepam, which has been shown previously to interact with at least two different binding sites in 122 receptors (Walters, Hadley et al. 2000), not only enhanced GABA-induced current via the benzodiazepine binding site, but at concentrations above 10 M also inhibited this current by interacting with the +/- interface (Baur, Tan et al. 2008). We thus investigated whether flurazepam is causing its inhibiting effect at high concentrations via the CGS 9895 binding site at the 1+3- interface. Results indicated that flurazepam at 250 M was able to inhibit the GABA-stimulatory effect of 10 M CGS 9895 in 13 receptors from 413±25% to 167±28% (n=4, p<0.001). In dose-response experiments flurazepam exhibited no effects in 13 receptors up to 1 M. At higher concentrations, this compound dose dependently inhibited GABA EC3, indicating that at this receptor flurazepam at high concentrations behaves as a negative allosteric modulator (Fig. 18). This negative modulatory effect of flurazepam, however, could not be inhibited by covalently modifying residue 3Q64C or 1V211C using MTSEA-biotin (Fig. 18 (Fig. 18). The absence of inhibition of the negative allosteric effect of high concentrations of flurazepam by all these covalently modified residues seems to indicate that the negative
46
Results allosteric effect of flurazepam in 13 receptors is not mediated via the extracellular 1+3- interface.
Fig. 18 Inhibitory effect of flurazepam at 13receptor receptors is not affected by mutants at + or - side. Concentration-response curves of flurazepam on 13 (○), 13Q64C (▲), 13Q64C receptors plus MTSEA-biotin (∆), 1V211C3 (■), 1V211C3 receptors labelled by MTSEA-biotin (□). The experiments were performed 2- 7 times in different oocytes. Neither any mutant nor the covalent modification with MTSEA-biotin was able to reduce the effect of flurazepam.
5.6 CGS 9895 differentially modulates different receptor subtypes via its binding site at the +- interface
To investigate a possible receptor subtype-selectivity of CGS 9895, receptors containing the 3 and different subunits were investigated. As shown in Fig. 19A, CGS 9895 exhibited the highest stimulation of GABA- induced current in receptors composed of 13 subunits. In receptors composed of 23, 33, or 53 subunits, the effect of CGS 9895 for stimulating GABA-induced currents was only half of that for 13 receptors. Similar results were obtained in receptors composed of x32 subunits. At 10 M the effects of CGS 9895 on GABA-induced current in 132 receptors was approximately twice as high as that on receptors composed of 232, 332 or 532 subunits (Fig. 19C). Interestingly, however, in contrast to 132, 232 and 532 receptors, that could not be modulated at low CGS 9895 47
Results concentrations, receptors composed of 332 subunits could be weakly stimulated (up to 130% at 100nM) at nM concentrations, indicating that CGS 9895 is a weak positive allosteric modulator at 332 receptors at these concentrations. Since this effect could be inhibited by covalent labelling of 2M130C with MTSEA-biotin in 332M130C receptors (Fig. 19D), this weak positive modulatory effect was mediated via the benzodiazepine binding site. These data indicate that CGS 9895 is an antagonist at the benzodiazepine binding site of 132, 232, or 532 receptors, but a weak positive allosteric modulator at the benzodiazepine site of receptors composed of 332 subunits.
In other studies, the influence of the type of the beta subunit on the CGS 9895 stimulation of GABA-induced current was investigated. Results obtained indicated that this compound exhibits a comparable stimulation of GABA- induced currents in 13 and 12 receptors, but nearly no stimulation in 11 receptors (Fig. 19B). The lack of effect of CGS 9895 on 1-containing receptors is similar to that of several other GABA modulators, such as loreclezole, etomidate, or DMCM (Stevenson, Wingrove et al. 1995). Since their beta-selectivity can be modulated by a point mutation of the amino acid residue 290, that is an asparagin (Asn) in the 2 or 3 subunit and a serine (Ser) in the 1 subunit, we investigated whether the mutation 1S290N can enhance the effect of CGS 9895 on 11 receptors. As shown in Fig. 19B, 10 M CGS 9895 were able to modulate GABA-induced currents in 11 receptors containing the point mutation 1S290N to a similar extent as in 12 or 13 receptors. Since residue 1S290N is located within TM2 of this subunit and is far away from the putative binding site of CGS 9895 in the extracellular domain (distance of 3N290 to 1V211C or 3Q64C is approximately twice the length of MTSEA- biotin) these data suggest that N290 in this case probably is not involved in direct binding of this compound but in the transduction of its effect on GABA- induced chloride current.
48
Results
Fig. 19 CGS 9895 effects in GABAA receptors containing different or subunits. (A) Concentration-response curves of CGS 9895 on 13 (■), 23 (▲), 3 (▼) and 3 (●) receptors. The experiments were performed 3-11 times in different oocytes. The effects of 10 M CGS 9895 were significantly different for 13 receptors (p<0.01, one way ANOVA with Bonferroni´s post-test) (B) Concentration-response curves of CGS
9895 on 1 (▽), 1 (○), 3 (■), and 1S290N (▼) receptors. The experiments were performed 4-11 times in different oocytes. The effects of 10 M CGS 9895 were significantly different for 11, but not for the other receptors (p<0.001) (C) Concentration-response curves of CGS 9895 on 132 (■), 232 (▲), 32 (♦) and 32 (▼) receptors. The experiments were performed 3-10 times in different oocytes. The effects of CGS 9895 at 10 nM and 100 nM were significantly different at 32 as compared to the other receptors (p<0.05). The effects of 10 M CGS 9895 were significantly different only between 132 and 532 receptors (p<0.01, one way ANOVA with Bonferroni´s post-test) (D) Concentration-response curves of CGS 9895 on 32 (♦), 32M130C (■) and MTSEA-biotin labelled 32M130C (X) receptors. The effect of 10 and 100 nM CGS 9895 at 32 receptors was reduced at 32M130C and MTSEA-biotin labelled 32M130C receptors. The experiments were performed 4-6 times in different oocytes.
49
Discussion
6 DISCUSSION
Parts of the text are taken from (Ramerstorfer, Furtmuller et al. 2011)
6.1 Steric hindrance – a novel tool for the identification of drug binding sites
A variety of clinically important drugs are exerting their effects via GABAA receptors but additional drug binding sites might be present at these receptors (Ernst, Bruckner et al. 2005). To develop a tool for identifying possible drug binding sites at GABAA receptors, we established a steric hindrance approach. In a proof of principle we introduced a cysteine at the 2- side (2M130C) of the benzodiazepine binding pocket and demonstrated that by covalently labelling of this cysteine with MTSEA-biotin in 132M130C receptors the ability of diazepam to stimulate GABA-induced ion flux was drastically reduced without altering the effects of drugs interacting with other binding sites of these receptors. This actually was expected because MTSEA-biotin with its length of 12 Å (Teissere and Czajkowski 2001), when irreversibly bound to a cysteine located within a pocket cannot reach into another binding pocket. Due to its length and flexibility, however, this compound cannot be used for exactly localizing the binding site within the pocket.
We then used a similar approach for identifying drugs interacting with the 1+3- interface by introducing cysteines at the 1+ (1V211C) and 3- (3Q64C ) side and subsequently investigating whether covalent labelling of these cysteines by MTSEA-biotin could inhibit the effects of the drugs. The pyrazoloquinoline CGS 9895, which previously has been demonstrated to exhibit a high affinity for the benzodiazepine binding site of GABAA receptors (Yokoyama, Ritter et al. 1982; Brown and Martin 1984) was selected for these investigations because this drug was able to enhance GABA-induced currents in receptors containing 1 and 3 subunits, only. Several lines of evidence indicated that CGS 9895 exerts its GABA-enhancing action in 132 receptors
50
Discussion via the 1+3- interface. First, this drug at nM concentrations does not modulate GABA-induced currents, but completely prevents the action of diazepam in 132 receptors indicating that it acts as an antagonist at the benzodiazepine binding site (Bennett 1987). Second, only at M concentrations CGS 9895 was able to enhance GABA-induced currents in 132 receptors, but this effect could not be inhibited by the benzodiazepine site antagonist Ro15-1788, again indicating that it is not mediated via the benzodiazepine binding site. Third, CGS 9895 was able to elicit a comparable stimulation of GABA-induced currents in receptors composed of 13 or 132 subunits, but not in homo-oligomeric receptors composed of 3 subunits, only. Fourth, the inability of this drug of eliciting a current in the absence of GABA, and the GABA-enhancing effect of this drug argues against its interaction with the GABA binding sites located at the 1-3+ interfaces. Fifth, the GABA- enhancing effect of CGS 9895 could be partially inhibited by a steric hindrance mediated by MTSEA-biotin via cysteines introduced at the 1+ (1V211C) and 3- (3Q64C) side, and was even more inhibited when the steric hindrance was introduced via both the 1+ and the 3- side, finally supporting the conclusion that the respective binding site is located at the extracellular part of the 1+3- interface.
It has been shown previously that some benzodiazepine site ligands can also interact with GABAA receptors via binding sites different from the classical benzodiazepine site. For instance, Hauser (Hauser, Wetzel et al. 1997) demonstrated that [3H]flunitrazepam exhibits a high affinity binding site at 622 receptors, although this compound only at very high concentrations was able to displace [3H]Ro15-4513 binding from the respective benzodiazepine binding site. This high affinity flunitrazepam binding obviously elicited a negative modulatory effect at these receptors. In other experiments, it was demonstrated that the negative allosteric modulator [3H]Ro15-4513 exhibited a high affinity binding to 4/63 GABAA receptors that could be displaced by several other benzodiazepine site ligands (Hanchar, Chutsrinopkun et al. 2006). Finally, (Walters, Hadley et al. 2000) reported that in 122 receptors diazepam at
GABA EC3 not only enhanced GABA-induced currents at a nM concentration
51
Discussion via the benzodiazepine binding site, but also exhibited an additional stimulation of GABA-induced currents at M concentration which in contrast to the nM component could not be inhibited by the benzodiazepine antagonist flumazenil and was also present in 12 receptors. This site was possibly located within the membrane-spanning region of the receptor because it could be eliminated by introducing three point mutations into this region. These authors also demonstrated that flurazepam at high M concentrations produced an inhibitory effect at 122 receptors and speculated that this might reflect a desensitization of the GABA current. Finally, (Baur, Tan et al. 2008) provided some evidence that the site mediating the inhibitory action of flurazepam might be located at the extracellular 1+2- interface of GABAA receptors. Here we demonstrated that flurazepam could inhibit the GABA-enhancing effect of CGS 9895 in 13 receptors. Actually, flurazepam at high M concentrations acted as a negative allosteric modulator at 13 receptors, but this effect could not be inhibited by covalently modifying 1V211C, 1S204C, or 3Q64C, by MTSEA- biotin, suggesting that this flurazepam effect was mediated via a site different from that of CGS 9895 in 13 receptors. This finding and the multiple binding sites possibly present at GABAA receptors (Ernst, Bruckner et al. 2005) underscore the importance of the present approach providing a tool for localizing individual drug binding sites in GABAA receptors.
6.2 The anxiolytic actions of CGS 9895 seem to be mediated via the benzodiazepine binding site of GABAA receptors containing 3 subunits.
CGS 9895 has been demonstrated previously to be a potent ligand for the benzodiazepine binding site of GABAA receptors. In vivo it elicited its anti- anxiety effect at a dose comparable to that of diazepam (Bennett 1987). The anxiolytic effect of CGS 9895 thus cannot have been elicited via the M effect of this compound at 132 receptors. We therefore investigated the effects of
CGS 9895 on various other GABAA receptor subtypes. Results indicated that CGS 9895 at nM concentrations exhibited a weak positive allosteric effect on
52
Discussion
332 receptors that could be completely inhibited by MTSEA-biotin in 332M130C receptors, suggesting that it was mediated via the benzodiazepine binding site of these receptors. This effect at nM concentrations could not be observed in 132, 232, or 532 receptors, supporting the assumption that the anxiolytic effect of this compound is mediated via 332 receptors. The absence of muscle relaxant and sedative as well as locomotor effects of CGS 9895 (Brown and Martin 1984; Bennett 1987) can be explained by the absence of effects at GABAA receptors containing 1 or 5 subunits at low concentrations. In addition, the observation that CGS 9895 is able to antagonize the anticonvulsant (Brown, Martin et al. 1984) or muscle in- coordination effects of diazepam in the rotarod test (Bennett 1987) at low concentrations is consistent with the antagonistic effects of this compound on the action of diazepam on 132 receptors. On the other hand, CGS 9895 at 100 mg/kg p.o. caused a 30% protection against the pentylenetetrazole induced convulsions in the rat (Bennett 1987). At such concentrations plasma levels of CGS 9895 between 20-50 M were reached and thus, this effect could also have been generated via the effects of this compound elicited at M concentrations on 132 receptors.
6.3 Drugs interacting with the +- interface are candidates for a novel GABAA receptor pharmacology
Although most of the actions of CGS 9895 at low drug concentration seem to be mediated via the benzodiazepine binding site of GABAA receptors, drugs interacting via the extracellular 1+3- interface should exhibit highly interesting properties. Judged by the observation that CGS 9895 even at high concentrations can only enhance GABA-induced currents but not directly activate these receptors, drugs acting via the +- interface will only have GABA-modulatory properties such as the benzodiazepines. In contrast to the benzodiazepines, however, these drugs will interact with receptors composed of , , , and subunits and should thus exhibit a much broader action than benzodiazepines which can only interact with 2
53
Discussion receptors and to a lower extent to 1 or 3 receptors. Drugs acting via the +- interface at receptors containing the less abundant subunits , , and might even exhibit some novel effects so far not seen with benzodiazepine site ligands, since these subunits not only have a distinct regional and cellular distribution, but might also be involved in specific behavioural effects, that can now be specifically modulated.
Drugs affecting a broader range of different receptor subtypes than benzodiazepine site ligands might be especially important for the treatment of epilepsy. Although benzodiazepines are known to be excellent anticonvulsants, they are also known for tolerance development, which is a major limitation for a long time application. This might be due to the benzodiazepine-induced uncoupling of the GABA and benzodiazepine site of GABAA receptors possibly caused by phosphorylation (Ali and Olsen 2001). Such a mechanism presumably would not be possible for drugs interacting at the +- interface, since and subunits are essential for forming the GABA binding site and any phosphorylation of these subunits would also influence the ability of GABA to activate the channel. Thus, even after drug mediated down-regulation of receptors, drugs would still be able to modulate the remaining receptors. Hence, it can be expected that drugs active via the +- interface would be feasible for a long-term treatment of epilepsy (Sieghart, Ramerstorfer et al. 2011).
In addition, these drugs also will exhibit receptor subtype-selective actions depending on the exact or subunit type forming the binding site and thus dramatically expand the pharmacology of GABAA receptor subtypes and our ability to modulate specific receptor subtypes, such as xy, xy, xy, xy, xy or xy. This conclusion is supported by the present results indicating a difference in the ability of CGS 9895 for stimulating GABA-induced currents on xy receptors containing different or subunits. However, so far we are not able to selectively address just one specific subset of these receptors. Although CGS 9895 has preferences for specific subunits like the 1, 2 and 3, receptors containing other subunits will be modulated as well.
CGS 9895 is the first compound unequivocally identified to modulate GABAA receptors via the extracellular 1+3- interface. Although its potency for 54
Discussion interacting with this site is relatively low, this compound now can be used as a screening tool for the identification of other possible candidates interacting with this binding site. This approach is especially important for the identification of possible antagonists at this binding site. In the absence of a direct effect of such compounds at GABAA receptors, at present they only can be detected by their inhibition of the effects of CGS 9895. In addition, using the cysteine mutations 1V211C, 1S204C, and 3Q64C, and their modification by MTSEA-biotin, compounds can now be identified that cause a positive or negative modulation of GABAA receptors via this binding site and that might lead to the development of clinically important drugs with a different spectrum of action.
55
Abbreviations
7 ABBREVIATIONS
5 HT3 5-hydroxytryptamine (serine)
AChBP acetylcholine binding protein
ATP adenosine tri-phosphate
BZ benzodiazepine cDNA complementary DNA
CNS central nervous system cRNA complementary RNA
DEPC diethylpyrocarbonate
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
EC3 concentration eliciting 3% of maximum effect
EC20 concentration eliciting 20% of maximum effect
EC50 half maximal effective concentration
EtOH ethanol
GABA γ-aminobutyric acid
Gly glycine
HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
LGIC ligand-gated ion channels mRNA messenger RNA
MTSEA N-Biotinylaminoethyl Methanethiosulfonate
NaAc sodium acetate (3M, pH=5.5, Ambion)
56
Abbreviations nAChR nicotinic acetylcholine receptor nH Hill coefficient
PCR polymerase chain reaction
RNA ribonucleic acid
RT room temperature
SEM standard error of the mean
TE Tris - EDTA
TM transmembrane domain
57
References
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Acknowledgement
9 ACKNOWLEDGEMENT
I want to thank Prof. Werner Sieghart for his guidance and constant support during my thesis. I have profited tremendously from his excellent scientific knowledge.
I also want to thank Margot Ernst for her supervision and helpful discussions in which she was always able to clarify the mysteries of protein structure.
Furthermore, I would like to thank Roman Furtmüller, who introduced me into the field of electrophysiology and who showed me the multiplicity of troubles that can occur while measuring, and how to deal with them.
In addition, I want to acknowledge all my colleagues of the department. All of them together were necessary to establish this wonderful atmosphere at this department which made working here so much fun.
I also want to thank Kathi Kefeder, my parents and family who always believed in me, no matter how long it lasted.
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Introduction
10 PUBLICATIONS
Sieghart W, Ramerstorfer J, Sarto-Jackson I, Varagic Z, Ernst M. (2011). “A novel GABA(A) receptor pharmacology: drugs interacting with the α+β- interface.” Br J Pharmacol. doi: 10.1111/j.1476-5381.2011.01779.x.
Tretter V, Kerschner B, Milenkovic I, Ramsden SL, Ramerstorfer J, Saiepour L, Maric HM, Moss SJ, Schindelin H, Harvey RJ, Sieghart W, Harvey K (2011). “Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin.“ J Biol Chem.;286(43):37702-11.
Ramerstorfer J, Furtmüller R, Sarto-Jackson I, Varagic Z, Sieghart W, Ernst M (2011). “The GABAA receptor alpha+beta- interface: a novel target for subtype selective drugs.“ J Neurosci. ;31(3):870-7.
Fischer BD, Licata SC, Edwankar RV, Wang ZJ, Huang S, He X, Yu J, Zhou H, Johnson EM Jr, Cook JM, Furtmüller R, Ramerstorfer J, Sieghart W, Roth BL, Majumder S, Rowlett JK (2010). “Anxiolytic-like effects of 8-acetylene imidazobenzodiazepines in a rhesus monkey conflict procedure.“ Neuropharmacology.;59(7-8):612-8. Epub 2010 Aug 18.
Ramerstorfer J, Furtmüller R, Vogel E, Huck S, Sieghart W (2010). „The point mutation gamma 2F77I changes the potency and efficacy of benzodiazepine site ligands in different GABAA receptor subtypes.“ Eur J Pharmacol.;636(1- 3):18-27. Epub 2010 Mar 19.
Savić MM, Majumder S, Huang S, Edwankar RV, Furtmüller R, Joksimović S, Clayton T Sr, Ramerstorfer J, Milinković MM, Roth BL, Sieghart W, Cook JM (2010). “Novel positive allosteric modulators of GABAA receptors: do subtle differences in activity at alpha1 plus alpha5 versus alpha2 plus alpha3 subunits account for dissimilarities in behavioral effects in rats?“ Prog Neuropsychopharmacol Biol Psychiatry.;34(2):376-86. Epub 2010 Jan 13.
Rivas FM, Stables JP, Murphree L, Edwankar RV, Edwankar CR, Huang S, Jain HD, Zhou H, Majumder S, Sankar S, Roth BL, Ramerstorfer J, Furtmüller R, Sieghart W, Cook JM (2009). “Antiseizure activity of novel gamma- aminobutyric acid (A) receptor subtype-selective benzodiazepine analogues in mice and rat models.“ J Med Chem. ;52(7):1795-8.
Furtmueller R, Furtmueller B, Ramerstorfer J, Paladini AC, Wasowski C, Marder M, Huck S, Sieghart W (2008). “6,3'-Dinitroflavone is a low efficacy modulator of GABA(A) receptors.“ Eur J Pharmacol.;591(1-3):142-6. Epub 2008 Jul 2.
Sarto-Jackson I, Ramerstorfer J, Ernst M, Sieghart W (2006). “Identification of amino acid residues important for assembly of GABA receptor alpha1 and gamma2 subunits.“ J Neurochem.;96(4):983-95. Epub 2006 Jan 12. Curriculum Vitae
11 CURRICULUM VITAE
Persönliche Daten
Name Joachim Ramerstorfer
Geburtsdatum 24. 10. 1976
Staatsangehörigkeit Österreich
Ausbildung seit Oktober 2005 PhD Studium am Zentrum für Hirnforschung, Abteilung Biochemie und Molekularbiologie der Medizinischen Universität Wien
Juni 2005 Ablegung der Diplomprüfung
März 2004 – Juni 2005 Diplomarbeit am Zentrum für Hirnforschung, Abteilung Biochemie und Molekularbiologie des Nervensystems
Oktober 1996 Beginn mit dem Studium Chemie an der Universität Wien
Oktober 1995 – Mai 1996 Absolvierung des Wehrdienstes
Mai 1995 Ablegung der Reifeprüfung
1987 – 1995 Besuch des BRG in Bruck an der Leitha
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Curriculum Vitae
Präsentationen/ Teilnahmen:
11th Meeting of the Austrian Neuroscience Association (ANA); (2009, 09, 16- 18.)
Posterpräsentation (“Identification of a novel binding site at GABAA receptors”)
PhD-Symposium; (2006, 06, 22 – 23.)
Posterpräsentation (“Investigation of drug binding sites at the interface of
GABAA receptors”)
PhD-Symposium; (2007, 06, 21 – 22.)
Posterpräsentation (“Localizing binding sites at GABAA receptors”)
10th Meeting of the Austrian Neuroscience Association (ANA); (2007, 09, 21- 23.)
Posterpräsentation (“Localizing binding sites at GABAA receptors”)
PhD-Symposium; (2008, 05, 28 – 29.)
Posterpräsentation (“Localizing binding sites at GABAA receptors”)
Auszeichnungen:
2011, YSA Publication Award („The GABAA Receptor a+b- interface: A Novel Target for Subtype Selective Drugs”)
2007, 1. Posterpreis, PhD-Symposium
2006, 2. Posterpreis, PhD-Symposium
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