DIPLOMARBEIT Effects of the Atypical Antipsychotic Clozapine On

DIPLOMARBEIT

Eﬀects of the atypical antipsychotic clozapine on recombinantly expressed

GABAA receptor subtypes zur Erlangung des akademischen Grades Dr. med. univ. ausgefuehrt am Zentrum fuer Hirnforschung unter der Anleitung von Prof. Dr. Margot Ernst, PhD

Luca Leandro Silva Pita

05/2018 Contents

0.1 Acknowledgements ...... i 0.2 Abbreviations ...... ii 0.3 Abstract ...... iv 0.4 Abstract [german] ...... v

1 Introduction 1 1.1 GABA Metabolism ...... 2 1.2 GABA Transport ...... 3

1.3 GABAA Receptors ...... 5 1.3.1 Structure and Distribution ...... 5 1.3.2 Drug-receptor Interactions ...... 12

1.3.3 Endogenous GABAA Receptor Ligands ...... 13 1.3.3.1 GABA ...... 13 1.3.3.2 Other Endogenous Ligands ...... 14

1.3.4 Exogenous GABAA Receptor Ligands ...... 15 1.3.4.1 Benzodiazepines ...... 15 1.3.4.2 Anesthetics ...... 17

1.3.4.3 Other GABAA Receptor Ligands ...... 20

1.4 GABAB Receptors ...... 22 1.5 Schizophrenia ...... 25 1.5.1 Epidemiology ...... 26 1.5.2 Pathomechanism ...... 26 1.5.3 Pharmacological Management ...... 27 1.6 Clozapine ...... 29

2 Materials and Methods 32 2.1 Two-Electrode Voltage Clamp ...... 32 2.1.1 Xaenopus laevis oocytes ...... 32 2.1.2 Preparation of oocytes ...... 32 2.1.3 Electrophysiological recordings ...... 34

3 Results 36

3.1 α1β2 ...... 37

3.2 α2β3 ...... 38

3.3 α1β2γ2 ...... 39

3.4 α2β3γ1 ...... 40

3.5 α2β3γ2 ...... 41 3.6 Summary ...... 42

4 Discussion 44 4.1 Exploring a possible intra-subunit binding pocket ...... 44 4.2 Agranulocytosis ...... 49

5 Summary and Outlook 50

List of tables 51

List of ﬁgures 53

Bibliography 55 0.1 Acknowledgements

I wish to thank, ﬁrst and foremost, Margot Ernst for the generous integration into her research group. Her encouraging way to allow for sovereign pursuit of emerging interests while providing a reliable framework with continuous, knowledgeable and heartfelt support promotes the formation of new ideas. I feel privileged having worked with someone who radiates true curiousity and dedication about her ﬁeld. Moreover I want to share the credit of this work with everyone I encoun- tered during my time at the CBR - thank you for interesting conversations, fun times and emerging friendships.

i 0.2 Abbreviations

2AG ...... 2-arachidonylglycerol

THDOC ...... 3 α5α-tetrahydrodeoxycorticosterone

DHP ...... 5 α-dihydroprogesterone

AMPA ...... α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

GABA ...... γ-aminobutyric acid

BBB ...... blood brain barrier

BZD ...... benzodiazepines

CNS ...... central nervous system

CLZ ...... clozapine

D2 ...... dopamine D2 receptor

DZP ...... diazepam

DMSO ...... dimethylsulfoxide

EC ...... eﬀective concentration

ECD ...... extracellular domain

FMZ ...... ﬂumazenil

GPCR ...... G protein-coupled receptor

GAT ...... GABA transporter

GAD ...... glutamate decarboxylase

GlyR ...... glycine receptor

ii ICD ...... intracellular domain

MDD ...... major depressive disorder mRNA ...... messenger ribonucleic acid

NMDAR ...... N-methyl-D-aspartate receptor

PKA ...... protein kinase A

RT-PCR ...... real-time polymerase chain reaction

SLC6 ...... solute carrier family 6

TMD ...... transmembrane domain

TEV ...... two-electrode voltage clamp

VFTD ...... venus ﬂy-trap domain

VGAT ...... vesicular GABA transporter

VGCC ...... voltage-gated calcium channel

WHO ...... World Health Organization

XR ...... xaenopus ringer

iii 0.3 Abstract

Clozapine (CLZ) is a tetracyclic compound with outstanding clinical ef- fectiveness in the treatment of psychotic symptoms even in patients resistant to high doses of typical neuroleptics [Naber and Lambert, 2009,Attard and Taylor, 2012]. While CLZ has been shown to interact with multiple targets [Ashby and Wang, 1996, Michel and Trudeau, 2000, Korpi et al.,

1995, Khokhar et al., 2018], its action at subtypes of the GABAA receptor family presents a unique feature when compared to the typical antipsychotic haloperidol [Korpi et al., 1995]. Treatment with CLZ is limited by its serious and potentially fatal side eﬀects, which include increased seizure suscepti- bility and agranulocytosis [Pacia and Devinsky, 1994, Alvir et al., 1993].

As GABAergic interneurons form an undisputed core component of cor- ticolimbic circuity, defects in GABA transmission have become a central element in a number of models for psychiatric disease such as schizophrenia and bipolar disorder [Benes and Berretta, 2001].

In this work we studied CLZ’s effects on five different recombinantly expressed GABAA receptor subtypes in electrophysiological experiments on Xenopus laevis oocytes. We show extensive negative allosteric modulation of GABA elicted currents at micromolar CLZ concentrations in all studied subtypes. Our results confirm that the existence of a γ subunit is no premise for CLZ sensitivity. The maximum mean inhibition at 100umol CLZ is significantly higher in α2 containing assemblies (α2β3γ2, α2β3γ1 and α2β3) compared to α1 containing assemblies (α1β2 and α1β2γ2).

In light of hints gained from structural models of analog proteins, we explore a tentative intra-subunit binding site candidate for CLZ and related molecules. Our ﬁndings prompt for a deeper examination of a possible link between some of CLZs clinical eﬀects and its negative modulatory action on

GABAA receptors.

iv 0.4 Abstract [german]

Clozapin (CLZ) ist ein tetracyclischer Wirkstoﬀ mit herausragender klinis- cher Wirksamkeit in der Behandlung psychotischer Symptome in Patienten, die nicht auf typische Neuroleptika ansprechen [Attard and Taylor, 2012]. Waehrend der Wirkmechanismus von CLZ unzureichend geklaert ist, kon- nten Interaktionen mit verschiedenen Zielmolekuelen experimentell gezeigt werden [Ashby and Wang, 1996, Michel and Trudeau, 2000, Korpi et al.,

1995]. Die Beeinflussung von Stroemen in Subtypen der GABAA Rezeptor- familie stellt eine einzigartige Eigenschaft im Vergleich zum typischen Neu- roleptikum Haloperidol dar [Korpi et al., 1995]. Die Behandlung mit CLZ ist begrenzt durch ihre schweren und potentiell toelichen Nebenwirkungen [Pa- cia and Devinsky, 1994, Alvir et al., 1993]. In dieser Arbeit wurde die Wirkung von CLZ auf fuenf verschiedene rekombinant exprimierte GABAA Rezeptor Subtypen in elektrophysiologis- chen Experimenten an Xaenopus laevis Oozyten untersucht. In allen un- tersuchten Subtypen zeigen wir eine ausgepraegte Reduktion von GABA induzierten Stroemen durch mikromolare CLZ Konzentrationen. Die Ergeb- nisse zeigen, dass die γ Untereinheit keine Vorraussetzung fuer CLZ Sensitiv- itaet ist. Die maximale mittlere Inhibition bei 100umol CLZ ist signifikant hoeher in α2 beinhaltenden Subtypen (α2β3γ2, α2β3γ1 und α2β3) verglichen mit α1 beinhaltenden Subtypen (α1β2 und α1β2γ2). Im Licht der Erkenntnisse aus Vergleichen mit Strukturmodellen analoger Proteine schlagen wir eine intra-subunit Bindestelle als Kandidat fuer die Vermittlung der negativ modulatorischen Effekte von CLZ vor. Die Ergeb- nisse regen zu einer tieferen Auseinandersetzung mit dem potentiellen Zusam- menhang zwischen negativ modulatorischen Effekten auf GABAA Rezep- toren und einigen von CLZs klinischen Effekten an.

v 1. Introduction

The human CNS is a network comprised of approximately 100 billion neural cells connected through a large number of synaptic interfaces. These synapses allow for phasic signal transmission between cells through neurotransmitter release. Neurotransmitters trigger excitatory (depolarizing) or inhibitory (hyperpolarizing) changes in the postsynaptic cell as a result of interactions with target molecules (receptors). Neurotransmitter release and action also occurs at extrasynaptic locations, leading to an overall change in the system’s excitability; a principle referred to as tonic signaling. Once the net depolarization of a neural cell reaches a certain threshold, an action potential is triggered, ultimately enabling subsequent signal transmission.

In healthy brains, excitation and inhibition are ﬁnely attuned, con- tributing to an environment promotive for information ﬂow and cellular health [Wolfe et al., 2010,Schonfeld-Dado and Segal, 2009]. A disruption of this equilibrium can be observed in a multitude of neuropsychiatric disorders, including schizophrenia and epilepsy [Schnitzler and Gross, 2005].

While glutamic acid constitutes the principal excitatory transmitter, γ- aminobutyric acid (GABA) and glycine carry out inhibitory neurotrans- mission in mammals. GABA transmission occurs at an estimated 30% of mammalian synapses, making it a principle signalling mechanism. Its action goes beyond simple inhibition, as GABA can also exhibit excitatory, shunting and modulatory eﬀects [Ernst et al., 2018]. Exemplary, GABAer- gic interneurons are involved in the integration of synaptic inputs through

1 oscillatory modulation of membrane potentials [Schnitzler and Gross, 2005]. The early hypothesized purely inhibitory nature of GABA was first chal- lenged by observation of depolarizing effects in immature neurons [Ben-Ari et al., 1989]. Later these effects were found to be present and important even in the adult CNS [Spitzer, 2010].

Besides the GABA systems fundamental physiological roles, GABA receptors constitute molecular targets of essential and widely employed drugs such as benzodiazepines and anesthetics. Research into the GABA system has become a fundamental focus, revealing complex arrangements heterogeneous in structure and function. Findings suggest major parts in the formation of disease [Benes and Berretta, 2001, Brickley and Mody, 2012] and promising potential for novel pharmacological intervention is dis- cussed [Rudolph and Mohler, 2014, Charych et al., 2009].

This work investigates the role of a subset of GABAA receptor family members as molecular targets of the established atypical antipsychotic drug clozapine.

1.1 GABA Metabolism

GABA is a non proteinogenic amino acid formed by decarboxylation of glutamic acid. The reaction uses peridoxal phosphate and is catalyzed by glutamate decarboxylase (GAD).

Figure 1.1: Simpliﬁed decarboxylation of glutamic acid yielding GABA

2 In mammals, two variants of GAD (GAD67 and GAD65) are encoded in seperate genes. The transcription of GAD67 starts during early embryonic development and shows an even intracellular distribution. While experiments with knockout mice reveal the importance of both isoforms during early developmental stages, deﬁcits are more severe in mice lacking GAD67 [Kakizaki et al., 2015]. Initial transcription of GAD65 takes place during later developmental stages and is localized primarily in nerve end- ings. Both forms of GAD appear to be expressed solely in cells that use GABA for synaptic signal transmission [Olsen RW, 1999]. Enzymatic breakdown of GABA exclusively takes place intracellularly under the presence of α–ketoglutarate and is catalyzed by GABA transami- nase. During this reaction, α–ketoglutarate is converted into glutamic acid.

Figure 1.2: Simpliﬁed transamination of GABA, yielding succinic semialdehyde

This closed-loop reaction process is referred to as the GABA shunt, responsible for synthesis, conservation and metabolism of GABA [Olsen RW, 1999].

1.2 GABA Transport

As breakdown of GABA is conﬁned to intracellular locations, clearance from the synaptic cleft relies on a group of functional proteins termed GABA transporters (GAT). GAT belong to a family of neurotransmitter:sodium symporters known as the solute carrier 6 family (SLC6) in humans [Scimemi, 2014]. The SLC6 consists of 20 members, of which six have been identiﬁed

3 Figure 1.3: GABA transporter stoichiometry

to transport GABA (GAT1, GAT2, GAT3, BGT1, CT1 and TauT). While all six accept GABA as a substrate and rely on the presence of chloride and sodium, substrate specificity and distribution differs among subtypes. As the transport direction follows the sodium gradient, the main mode is inward. Labeling studies show a widespread expression of GAT1 and GAT3 in the rat CNS, whereas GAT2 expression is confined to the leptomeninges. GAT1 reaches its highest levels of expression in hippocampal areas and the olfactory bulb, while GAT3 is mainly found in the olfactory bulb, thalamus and hippothalamus. The cellular and sub-cellular distribution also varies pertinently among brain regions, suggesting specific roles in re-uptake and uptake [Scimemi, 2014]. Figure 1.4

Before its release, GABA is sequestered into vesicles in the presynaptic neuron. Uptake of GABA as well as glycine into vesicles is facilitated by the vesicular GABA transporter (VGAT). A reversible interaction between GAD65 and VGAT has been shown, indicating that presynaptic synthesis from glutamate and vesicular uptake is a highly coupled process. [Jin et al., 2003]

GABA’s vital role during embryonic development is further reinforced by knockout experiments with GAD67, GAD65 and VGAT. Knockout of VGAT

4 Figure 1.4: A) Schematized morphology of cortical pyramidal neuron (top) and of the distribution of GAT1 (green) and GAT3 (blue) at synaptic contacts onto these cells (bottom). B),C) As in A, for Purkinje and thalamic relay neurons. [Scimemi, 2014] results in the most severe embryonic malformations due to near complete loss of signaling ability [Kakizaki et al., 2015].

1.3 GABAA Receptors

1.3.1 Structure and Distribution

GABAA receptors form a heterogeneous family of membrane bound, ligand gated anion channels formed by an arrangement of five subunits. Each subunit’s extracellular domain (ECD) is composed of a large N-terminal and a smaller C-terminal, connected by four α-helical transmembrane domains (TMD) and an intracellular domain (ICD) of variable length between TM3 and TM4 [Schofield et al., 1987]. 19 different subunit genes (six α, three β, three γ, three ρ, one δ, one , one θ and one π) have been identified in mammals, located on seven different chromosomes (1, 3, 4, 5, 6, 15, X)

5 in humans [Sigel and Steinmann, 2012]. Amino acid sequence overlap is greatest within a single subunit class, whereas it decreases when comparing sequences across subunit classes [Ernst et al., 2018]. The phylogenetic tree shown in Figure 1.5 depicts degrees of sequence similarity as well as gene location [Ernst et al., 2018].

Figure 1.5: The phylogenetic tree depicts the degree of similarity (based on % sequence similarity) of the 19 GABR genes. In humans, Chromosome 5 harbours a gene cluster with GABRa1, GABRa6, GABRb2, GABRg2 and GABRp; Chro- mosome 4 harbours a homologous cluster with GABRa2, GABRa4, GABRb1 and GABRg1; on Chromosome 15, we ﬁnd GABRa5, GABRb3 and GABRg3, the X- Chromosome contains GABRa3, GABRq and GABRe. [Ernst et al., 2018]

While we lack complete information on occurence and distribution of speciﬁc pentameric assemblies in human brains, most prevalent GABAA receptors consist of two α, two β and one γ subunit [Sieghart, 2015] in alternating order [Tretter et al., 1997]. Detection of subunit messenger ribunucleic acid (mRNA) by real-time polymerase chain reaction (RT-PCR), as well as immunocytological charac- terizations on adult rat brain slices have provided extensive information

6 on estimated subunit distribution among brain regions [H¨ortnaglet al., 2013,Pirker et al., 2000,M¨ohleret al., 2002]. The knowledge obtained from these studies, as well as electron microscopy [Nayeem et al., 1994], homology models of the extracellular domain and experiments with concatenated subunits allow for likely estimates about major existing asseblies: α1β2γ2

(60%), α2β3γ2 (15-20%), α3βnγ2 (10-15%), α4βnγ/δ (5%), α5β2γ2 (<5%) and α6β2/3γ2 (<5%) [M¨ohleret al., 2002, Olsen and Sieghart, 2008]. For a recent, comprehensive summary of proposed, high likelyhood and tentative receptor assemblies see Table 1.1 published by [Ernst et al., 2018].

The α1 subunit is unequivocally dominant in prevalence and ubiquity in mammalian brains, whereas expression of other α subunits appears to be more conﬁned to certain brain regions [Pirker et al., 2000]. High level expression of the α6 subunit, for instance, is clearly restricted to the cere- bellum in immunochemical mouse brain mappings [H¨ortnagl et al., 2013]. (Figure 1.6) The observed heterogeneity in mRNA expression appears to be strongest during early embryonic development opposed to the adult stage [Ernst et al., 2018].

While conventional αβγ containing assemblies are mainly involved in direct synaptic transmission, δ as well as α4-6 containing expression patterns were often identiﬁed in extrasynaptic locations [Wisden et al., 1992, Wang, 2011]. A tonic form of inhibition mediated by δ containing subtypes in cerebellar regions was ﬁrst shown in-vivo by Chadderton et al. [Chadderton et al., 2004]. Opposed to the fast, transmitter-release dependent inhibition seen at synapses, extrasynaptic GABAA receptors enable a tonic form of inhibition governed by the continuous presence of ligands [Kullmann et al., 2005].

A three dimensional protein structure has been resolved via x-ray crys- tallography for the ECD and TMD of a β3 homopentameric assembly by Miller et al. [Miller and Aricescu, 2014]. Interfaces between two neighbour-

7 Table 1.1: Receptor subunits and compositions proposed to exist (blue), or assumed to exist with high probability (red) or tentative (green). Note that the tentative arrangements are exemplary mentions and not complete. Modiﬁed from [Ernst et al., 2018]

Gene/subunit Receptor composition

GABRa1 α1 α1βxγ2, α1β2δ, α1βxδ, α1αxβxγx, α1αxβxδ, α1βx

GABRa2 α2 α2βxγ2, α2β1γ1, α2αxβxγx

GABRa3 α3 α3βxγ2, α3βx, α3βxθ

GABRa4 α4 α4βxγ2, α4βxδ, α4αxβxγx, α4αxβx

GABRa5 α5 α5βxγ2, α5αxβxγx

GABRa6 α6 α6βxγ2, α6α1βxγ2, α6βxδ, α6α1βxδ

GABRb1 β1 αxβ1γ2, α1β1δ, α4β1δ, α2β1γ1, αxαxβ1βx

GABRb2 β2 α1β2γ2, αxβ2γ2, α4,6β2δ, α1β2δ, αxαxβ2βx

GABRb3 β3 α1β3γ2, αxβ3γ2, α4,6β3δ, αxαxβ3βx

GABRg1 γ1 αxβxγ1

GABRg2 γ2 αxβxγ2, αxβxγ2

GABRg3 γ3 αxβxγ3

GABRd δ α1βxδ, α1β2δ, α4βxδ, α6βxδ, α1α6βxδ

GABRe α3βx, α3θ

GABRq θ α3βxθ GABRp π nothing known GABRr1 ρ1 ρ subunits are thought to coassemble with each other...

GABRr2 ρ2 ...(e.g. ρ1 homopentamers, ργ2)... GABRr3 ρ3 ...see above

ing subunits form binding sites for several known ligands. The space between two extracellular domains contains several accessible pockets, which

8 Figure 1.6: Distribution of GABAA receptor mRNAs (α1-6,β1-3,γ1-2 and δ) in horizontal sections of the mouse brain at the level of Bregma -2.56 to -2.36 mm. Scale bar = 2 mm [H¨ortnaglet al., 2013]

harbour binding sites that serve for agonist recognition and allosteric modulation [Ernst et al., 2018].

9 Figure 1.7: Topology of the typical GABAA receptor subunit polypeptides, based on 4COF and 4PIR crystal structures. The dashed line indicates the ICD fragment that is highly variable in structure sequence and legth between subtypes. The cys-loop is shown in yellow in the otherwise lilac ECD, ECD interface binding site- forming segments A-G are indicated with green capital letters. The junction zone loops and the termini are colored in grey, the four TMD helices and the HX helix, which in homologous cation channels is located in the TMD as well, are coloured in red, while the ICD comprising structures are coloured green. [Ernst et al., 2018]

The extracellular domain comprises a beta-stranded fold, where seven segments (traditionally called ”loops” A-G) form the two interfaces where each subunit is in contact with its neighbors (the principal- or plus- side, and the complementary- or minus- side, respectively). (Figure 1.7 and Figure 1.8)

10 Figure 1.8: This ﬁgure shows a side view of the interface between the β3 (yellow ribbon) and α2 (grey ribbon) subunits in a homology model of an α2β3γ2 receptor. Extracellular segments or ”loops” are highlighted by color. The model is based on the available β3 homopentamer structure 4COF [Miller and Aricescu, 2014].

Based on the available structures of related proteins of the same super- family, homology models have been employed to identify and characterise putative binding sites for various ligands [Puthenkalam et al., 2016].

To date, a wide range of binding sites for exogenous ligands such as propofol, etomidate, barbiturates, benzodiazepines, avermectin, furosemide, clozapine, alcohol and some antidepressants have been described and partly conﬁrmed [Ernst et al., 2018, Sieghart, 2015, Squires and Saederup, 1998, Korpi et al., 1995].

11 Figure 1.9: Ten small ligand binding sites are found in atomic structures of

GABAA receptor homologs. The ﬁgure shows a side view of a superposition of available structural models listed in the original study. Two subunits of 4COF are shown in ribbon representation (gray). The ligands are depicted in space-ﬁlling representation. [Puthenkalam et al., 2016]

Opposed to the direct agonistic eﬀect of GABA, substances with allosteric activity modulate the receptors sensitivity to the eﬀects of GABA by inducing a conformational change.

1.3.2 Drug-receptor Interactions

This section serves the introduction of basic pharmacologic terminology re- garding drug-receptor interactions. Aﬃnity is a measure for the binding ability of a ligand to a site on the target molecule. It is descriptive of the inter-molecular forces to form a complex. Intrinsic activity describes the probability of an already formed ligand-receptor complex to result in a functional change.

12 Agonists are ligands capable of inducing a functional change in the receptor that corresponds to the main physiological ligand. (E.g. increased channel conductivity in case of GABAA receptors) Antagonists block or dampen the receptors response/state that can be triggered by agonists. In- verse agonists elict a functional change opposite to that of an agonist. The primary, physiological binding site on a receptor is referred to as orthosteric, while sites at other locations are termed allosteric. Binding at allosteric sites can induce conformational changes that directly affect receptor function or mediate modulation of other ligands effects. (Negative allosteric modulation (NAM) or positive allosteric modulation (PAM)). If two ligands have a common binding site, they are competetive, whereas allosteric effects are non-competitive.

1.3.3 Endogenous GABAA Receptor Ligands

1.3.3.1 GABA

A high affinity, extracellular, orthosteric agonist binding site of GABA was identified at the β(+)/α(-) interface present in the most abundant existing assemblies [Smith and Olsen, 1995]. Binding of GABA induces a conformational change, selectively allowing chloride and carbonate anions to pass through the receptors central pore. The HCO3-/Cl- permeability ratio is 0.2-0.4 [Ernst et al., 2018]. Most common receptors are composed of two α, two β and one γ subunit, thus presenting two β(+)/α(-) interfaces. A specific model of putative interactions between GABA and its binding site has been obtained by combining information available from structural models of related proteins from [Ernst et al., 2003, Bergmann et al., 2013]. (Fig- ure 1.8) Simultaneous binding of GABA on both sites greatly enhances the likelihood of channel opening [Baumann et al., 2003]. GABA’s action mode explains the inhibitory nature of postsynaptic GABAA receptors, as chloride equilibrium potential and gradient in the mature neuron lead to an influx of

13 chloride. Immature neurons lack the extent of chloride transporters neces- sary to generate such gradient, thus allowing conditions under which GABA has excitatory properties [Olsen and Sieghart, 2008]. This mechanism is not confined to the developing CNS, but plays a crucial role for the integration of newly born neurons in the adult nervous system [Spitzer, 2010]. Affinity of GABA to its site is highly pH dependent, making adjustments an im- perative prequesite for experimental characterization [Huang et al., 2004]. An additional, low affinity binding site for GABA was recently proposed at the extracellular β(+)/δ(-) interface in αβδ containing receptors [Lee et al., 2016].

Subunit composition unambiguously constitutes a determining factor for GABA sensitivity and therefore determines the physiological function of a receptor. [Olsen and Sieghart, 2009]

1.3.3.2 Other Endogenous Ligands

Apart from GABA, numerous other endogenous substances are known to interact with GABAA receptors. It was proposed that most, if not all, GABAA receptor ligands can interact with more than one binding site [Sieghart, 2015].

Histamine is a neurotransmitter primarily known for mediating inﬂam- matory response. Many additional central nervous functions exist and a his- taminergic model of treatment resistant schizophrenia has been suggested [Ito,

2004]. A direct agonistic eﬀect on β3 homopentamers [Hoerbelt et al., 2016] has been shown. Additionally, positive modulatory properties were observed in extrasynaptic and synaptic α4 containing receptors [Bianchi et al., 2011, Saras et al., 2008]. Based on the available structure of the β3 homopentamer, a putative binding site was described in a pocket located at the extracellular interface between two β3 subunits [Ernst et al., 2018].

14 The endocannabinoid 2-arachidonylglycerol (2AG) has been shown to interact in a modulatory way with β2 containing GABAA receptors [Sigel et al., 2011]. A novel binding site involving the β2 TM4 was suggested [Sigel et al., 2011]. Later docking studies strongly support the proposed endocannabinoid binding site localized between TM4 and TM3 [Baur et al., 2013]. Neurosteroids present a class of endogenous steroids interacting directly with GABAA receptors. Their effect is determined by the specific char- cateristics of the neurosteroid, as well as the brain region, type of neuron, receptor location and subunit composition. Pregnenolone, progesterone, 5α- dihydroprogesterone (DHP), allopregnanolone, adiol, androstane and 3α5α- tetrahydrodeoxycorticosterone (THDOC) were shown to exhibit positive modulatory effects, whereas sulfated and 3β-OH steroids posess negative allosteric properties [Wang, 2011]. Findings suggest that binding of neu- roactive steroids takes place at the lower TMD interface irrespective of the subunit type [Ernst et al., 2018]. Based on recent crystal structures of the

GLIC-GABAAR α1 chimera bound to pregnolone sulfate and THDOC, two TMD binding sites were described. [Laverty et al., 2017].

1.3.4 Exogenous GABAA Receptor Ligands

1.3.4.1 Benzodiazepines

Figure 1.10: Benzodiazepine structure

15 Benzodiazepines (BZD) present a group of GABAA receptor modulators introduced into clinical use in the 1960s. BZD have since then found widespread clinical adoption, constituting frequently prescribed and essential drugs valued for their anxiolytic, anticonvulsant, sedative and muscle re- laxant properties while maintaining favorable toxic proﬁles [Sieghart, 2015].

A high affinity binding site for the action of BZDs has been identified at the extracellular α(+)/γ(-) interface [Sieghart, 2015]. Binding of most BZD induces a conformational change that increases the affinity of GABA to its orthosteric binding site at the β(+)/α(-) interface. BZDs thus positively modulate GABAA receptors in the presence of GABA [Sieghart, 2015]. Ad- ditionally, an increase of channel opening frequency has been demonstrated in electrophysiological experiments [Bianchi et al., 2009]. There are ligands of the benzodiazepine binding site such as flumazenil (FMZ), that exhibit negative allosteric modulatory (NAM) effects, thus competetively blocking the effects of classical benzodiazepines. FMZ is clinically employed to an- tagonize unwanted or excessive BZD effects. This NAM mechanism might also be responsible for the risk of paradoxical effects of some BZDs under certain circumstances.

Classical 1,4-benzodiazepines such as diazepam, ﬂunitrazepam, temazepam and lorazepam act on receptors containing α1,2,3,5+/γ2,3- interfaces. Op- posed to that, the chemically distinct alternative ligand of the same binding site, zolpidem, displays relative selectivity for α1γ2 containing receptors [Ernst et al., 2018].

Advances in the understanding of subtypes and their specific function have fueled the promising investigation into potentially novel, selective agents with more specific therapeutic profiles [Sieghart et al., 2012, Rudolph and Mohler, 2014].

16 Figure 1.11: Etomidate

1.3.4.2 Anesthetics

General anesthetics present a group of drugs employed for their hypnotic, immobilizing, anesthetic and amnestic properties. While most anesthetic agents positively modulate GABAA receptors, etomidate constitutes a sub- stance with relatively speciﬁc action on GABAA receptors containing β2 or

β3 subunits [Uchida et al., 1995,Forman, 2011,Martin et al., 2009]. Etomi- date evokes potent amnestic effects at relatively low doses. This presents a prominent feature when compared to other anesthetic drugs. It was shown that etomidate has negative effects on the plasticity of glutamatergic excitatory transmission in murine hippocampal slices [Martin et al., 2009]. After experiments with null mutant mice, the amnestic effects can be correlated with α5 containing receptors [Martin et al., 2009]. This finding is compatible with the murine mRNA expression pattern of α5 subunits, which is markedly enhanced in the hippocampi. (Figure 1.6) An interaction and possible binding site for etomidate was identified at the β+/α- and β+/β- interfaces in the upper TMD [Chiara et al., 2012].

Barbiturates present a class of drugs that, opposed to the positive modulatory action of benzodiazepines, exhibits direct agonistic properties on

GABAA receptors even in the absence of GABA. Two binding sites have been proposed for phenobarbital at the α+/β- and γ+/β- interfaces of the upper TMD [Chiara et al., 2013].

17 Figure 1.12: Phenobarbital

Propofol is a lipid-soluble anesthetic approved for intravenous application. Due to its rapid-onset and short eﬀect duration, it is not only employed for induction and maintenance of general anesthesia but also utilized for procedural sedation.

Figure 1.13: Propofol

In the TMDs of the bacterial ion channel GLIC, a protein homologous to GABAA receptors, an intrasubunit pocket for propofol exists. Results from photolabeling studies however indicate, that the intra-subunit binding site is not present in GABAA receptors [Sieghart, 2015]. It was shown that propofol causes potentiation and direct activation via binding sites at the

TMD α+/β-, γ+/β- and β+/α- interfaces of GABAA receptors with little or no speciﬁc subunit interface selectivity [Sieghart, 2015]. An additional possible binding site has been proposed in the intracellular loop of α1β2 receptors, based on experiments with mutated receptors [Moraga-Cid et al., 2011].

18 Figure 1.14: Ketamine

Ketamine is a water soluble phencyclidine (PCP) derivative with two enantiomers: The S(+) isomer and the R(-) isomer. Since its introduction into clinical use in the 1970s, ketamine has sustained its role as an important intravenous general anesthetic and induction agent. Opposed to anesthetics of the opioid or barbiturate class, ketamine exhibits stimulatory effects on the cardiovascular system and bronchodilatation. Moreover, its newly described neuroprotective, antiinflammatory and antitumor effects implicate further potential benefits and applications [Kurdi et al., 2014]. Addition- ally, ketamine treatment for depression has recently gained popularity, as it provides rapid symptom relief compared to other antidepressant medications [Murrough et al., 2013].

While ketamines main anesthetic mechanism is likely caused by strong, non-competetive antagonism at N-methyl-D-aspartate receptors (NMDAR), interactions with a multitude of other receptors are evident. Observa- tions in mice reveal that the metabolite (2R,6R)-hydroxynorketamine might contribute to the antidepressant eﬀects by interaction with AMPA receptors [Zanos et al., 2016]. Another experiment with mice suggests the activation of GABAA receptors as a potential pathway for ketamines antide- pressive action [Rosa et al., 2016]. A potentiation of GABAergic conductance arising from α6-containing GABAA receptors was shown in dissociated granule neurons [Hevers et al., 2008]. Based on the bacterial GLIC crystal

19 structure bound to R-ketamine, a putative binding site was described at the extracellular interface between two subunits [Puthenkalam et al., 2016]. (Figure 1.9) Ketamine eﬀects entail a strong, psychoactive component, that is per- ceived as undesirable and intimidating by many patients. The dissociative and hallucinatory state caused by ketamine is used as a model for the study of positive, negative, and cognitive symptoms, dopaminergic and GABAer- gic dysfunction, age of onset, functional dysconnectivity, and abnormal cortical oscillations observed in acute schizophrenia [Frohlich and Van Horn, 2014].

Figure 1.15: Desﬂourane

Numerous inhalative anesthetics, such as desﬂourane, isoﬂourane, en-

ﬂourane, halothane, chloroform and ethanol positively modulate GABAA receptors. Acumulated evidence suggests that a binding pocket for these compounds exists within the four α-helices of the α subunit TMD [Sieghart, 2015].

1.3.4.3 Other GABAA Receptor Ligands

A vast variety of other compounds, many of which are naturally ocurring and plant derived, interact with GABAA receptors. Bicuculline, found in plants of the Corydalis species, as well as muscimol, isolated from Amanita muscaria, present alkaloids directly interacting via the GABA binding site [Johnston et al., 1968].

20 Many natural and synthetically derived flavonoids display modulatory effects via the high affinity BZD binding site [Baureithel et al., 1997]. Hispidulin presents a flavonoid with potent positive allosteric properties, exhibiting anticonvulsive effects in mice [Kavvadias et al., 2004]. Terpenes form a large, heterogenous class of compounds almost ubiqui- tous in plants. They are likely responsible for the therapeutic effects of many traditional, plant derived medicines. Exemplary, valerenic acid, present in plants of the Valeriana genus, mediates anxiolysis and sedation through allosteric modulation of GABAA receptors [Becker et al., 2014]. Currently, cannabinoids such as cannabidiol (CBD) and tetrahydrocannabi- nol (THC) are increasingly recognized, used and investigated for their broad therapeutic potential. Plant cannabinoids modulate GABAA receptors in differential and complex ways, possibly constituting a core pathway for some of their therapeutic effects. Several binding sites for small molecules and atoms such as Br-, Ba2+ and Br-ethanol have been identified [Puthenkalam et al., 2016]. Figure 1.9 Other known synthetic modulators include some tricyclic antidepressants such as amoxapine, several anticonvulsive drugs such as carbamazepine, the antiparasitic ivermectin, as well as the atypical antipsychotics chlorpromazin and clozapine [Squires and Saederup, 1998,Sieghart, 2015,Korpi et al., 1995].

21 1.4 GABAB Receptors

Opposed to the ionotropic nature of GABAA receptors, GABAB receptors present a family of G protein-coupled receptors (GPCRs). They are composed of two principal subunits, each formed by seven transmembrane domains and a large extracellular ’venus fly-trap domain’ (VFTD) that contains the ligand-binding site [Gassmann and Bettler, 2012]. The principal subunit compositions are GABAB1a,2 and GABAB1b,2. G-protein coupling occurs intracellularly at the GABAB2 subunit, whereas extracellular ligand binding takes place at the VFTD of GABAB1a or GABAB1b [Rondard et al., 2011]. Additionally, the auxiliary subunits KCTD8, KCTD12, KCTD12b and KCTD16 were identified, which posess modular structure and feature a con- served tetramerization T1 domain as well as one or two carboxy-terminal ’homology’ domains (H1 and H2) of unknown functions [Schwenk et al., 2010]. Located intracellularly, close to the G-protein binding site, these auxiliary subunits take great part in the receptors kinetic and pharmacological profile [Gassmann and Bettler, 2012].

22 Figure 1.16: a) GABAB1a and GABAB1b are principal subunit isoforms that diﬀer by the presence of two amino-terminal sushi domains in GABAB1a. Whereas these subunits contain the GABA binding site, GABAB2 subunits couple to the G protein. b) The principal subunits associate with the sequence-related auxiliary subunits KCTD8, KCTD12, KCTD12b or KCTD16. The T1 domains form homotetramers that bind to GABAB2. [Gassmann and Bettler, 2012]

2+ GABAB receptors in presynaptic locations inhibit Ca inﬂux via Gβγ release and inhibition of voltage-gated calcium channels (VGCCs). Addi- tionally Gβγ was shown to inhibit vesicle fusion and therefore spontaneous neurotransmitter release through direct interaction with SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) [Gassmann and Bettler, 2012]. Release of the larger Gα protein also reduces spontaneous neurotransmitter release through inhibition of adenylyl cyclase [Gassmann and Bettler, 2012].

23 Diﬀerent from its main eﬀectors in presynaptic cells, postsynaptic GABAB receptors mediate inhibition mainly through activation of G protein-coupled inwardly-rectifying potassium channels (GIRKs) via released Gβγ [Gassmann and Bettler, 2012]. Activated GIRK reduce neuronal excitability by gener- ating slow IPSPs [Gassmann and Bettler, 2012].

Figure 1.17: a) Downstream effectors of presynaptic GABAB receptors are depicted by arrows indicating the effect on target proteins, physiological roles are represented in the boxes. b) Downstream effectors of postsynaptic GABAB receptors are depicted by arrows indicating the effect on target proteins, physiological roles are represented in the boxes. [Gassmann and Bettler, 2012]

24 Additional downstream eﬀectors such as the potassium channel TREK2 and N-methyl-D-aspartate receptor (NMDAR) have been identiﬁed and are depicted in Figure 1.17 from [Gassmann and Bettler, 2012]. Downregu- lation of protein kinase A (PKA) as a result of adenylyl cyclase inhibition was shown to take part in gene regulation [Fukui et al., 2008].

Post mortem studies reveal decreased expression of GABAB receptors in bipolar disorder, major depressive disorder (MDD) and schizophrenia [Fatemi et al., 2011]. A signiﬁcant link between polymorphisms in the KCTD12 auxiliary subunit gene and risk of bipolar disorder was identiﬁed in a chinese population [Lee et al., 2011]. Knockout studies with mice support the hypothesis that KCTD12 plays a central role in mood regulation [Cathomas et al., 2015].

GABAB receptors have been proposed as novel drug targets for various neuropsychiatric disorders [Kumar et al., 2013].

1.5 Schizophrenia

The World Health Organization (WHO) classiﬁes schizophrenia in code F20 of the International Statistical Classiﬁcation of Diseases and Related Health Problems (ICD10).

Schizophrenia presents a spectrum of chronic syndromes characterized by severe deviations from ordinary perception and thought. Sense of con- sensual reality is greatly disturbed, impairing the ability to interact in mu- tually comprehensible ways. Delusions, hallucinations and unusual thought processing contribute to an overly intense and often threatening experience of reality. Additionally, a disruption of cognitive and aﬀective functioning contributes to the syndromes severely debilitating eﬀects on many aspects of life. Symptoms are typically divided into two categories, positive and negative. (Table 1.2)

25 Table 1.2: Schizophrenia symptom categories

positive negative

delusions aﬀective ﬂattening hallucinations alogia disorganized speech anhedonia disorganized behaviour avolition

1.5.1 Epidemiology

With a global prevalence between 0.5% and 1.6%, the syndrome poses a major global health challenge. Yearly incidence lies at 0.02%, greatly vary- ing among regions [Messias et al., 2007]. About 50% of cases present an acute initial onset, while the other half is preceded by a prodromal phase. Incidence peaks at an age range of 15-24 years for both sexes, but males have a 30%-40% increased lifetime incidence risk. Positive symptoms oc- cur close to the first hospitalization, while negative symptoms emerge on average five years later [Messias et al., 2007]. Schizophrenia is associated with a weighted average of 14.5 years of potential life lost [Hjorthøj et al., 2017]. Besides a markedly increased risk for suicide, comorbid conditions such as cardiovascular disease contribute to the significantly reduced life expectancy [Skalicky, 2017, Piotrowski et al., 2017].

1.5.2 Pathomechanism

Over past decades, extensive research has been conducted in order to eluci- date pathogenetic mechanisms of schizophrenia. While substantial advances lead to an improved management, identiﬁcation of distinct underlying pathways remains elusive. The most persistent and long-standing hypothesis is based on excessive dopaminergic signalling. It was reinforced by post

26 mortem findings, the resemblance between states evoked by dopaminergic, stimulant drugs and positive symptoms, as well as the antipsychotic efficacy of dopamine D2 blocking agents. The link between increased dopamine signalling and schizophrenia remains unequivocal, albeit not satisfactory in light of the current body of evidence. Significant familial coincidence, as high as 41% - 61% among identical twins, lead to extensive efforts to characterize the syndromes genetic makeup. While unequivocal and substantial, the genetic components of schizophrenia are inherited in complex and non-mendelian ways [Harrison, 2015], limiting distinct conclusions. See table 1.2 for select identified loci and genes associated with schizophrenia. Accumulated evidence suggests a complex, likely multicausal etiology involving several neurotransmitter systems beyond dopamine, such as glutamate, serotonin and GABA [Schwartz et al., 2012,Balu et al., 2013,Charych et al., 2009, Benes and Berretta, 2001, Verrall et al., 2010, Barkus et al., 2014]. In efforts to pharmacologically address some of schizophrenias cognitive symptoms, evidence suggests that α2 containing GABAA receptor assemblies might play a special role as a target [Charych et al., 2009]. Fol- lowing this lead, we focused on α2 containing assemblies for characterization of CLZ effects.

1.5.3 Pharmacological Management

First generation antipsychotic drugs are characterized by strong antagonistic effects on dopamine D2 receptors. As a consequence of strong D2 blockade, these typical antipsychotics evoke extrapyramidal side effects [Meltzer, 2013]. Subsequently, new antipsychotic compounds were discovered that proved to be effective even with relatively weak dopamine D2 blockade. The action mode of these atypical antipsychotics remains largely elusive [Khokhar et al., 2018].

27 Table 1.3: Select loci and genes showing association with schizophrenia. (Modiﬁed from [Harrison, 2015])

Locus Implicated Name of Notes Reference to gene gene gene/product biology

12p13.33 CACNA1C L-type calcium Important in neuronal function. Muta- [Bhat et al., 2012] channel α sub- tions cause Timothy syndrome and Bru- unit, type 1c gada syndrome.

12q24.11 DAO D-amino acid oxi- Enzyme which degrades the NMDA re- [Verrall et al., 2010] dase ceptor co-agonist D-serine. Expression and activity increased in schizophrenia. Not GWAS signiﬁcant.

1q42.2 DISC1 Disrupted in Identified in a large Scottish pedigree [Brandon and Sawa, schizophrenia-1 with a chromosome 1:11 translocation. 2011] A multifunctional scaffolding protein. Not GWAS significant.

11q23.2 DRD2 Dopamine D2 re- Long known to be the key target of an- [Beaulieu and Gainet- ceptor tipsychotic drugs, GWAS data now in- dinov, 2011] dicate that the DRD2 gene may play a role in schizophrenia.

2q33-34 ERBB4 Receptor tyrosine Receptor for neuregulin 1 and some [Mei and Xiong, 2008] kinase erbB4 other ligands. Mutations can cause can- cers. Not GWAS signiﬁcant.

5q33.2 GRIA1 AMPA recep- The subunit inﬂuences properties of the [Barkus et al., 2014] tor subunit 1 AMPA receptor, and aﬀects synaptic (GluA1; GluR1) plasticity and behaviour.

16p13.2 GRIN2A NMDA recep- The subunit inﬂuences properties of the [Paoletti et al., 2013] tor subunit 2A NMDAR, including synaptic localisation (GluN2A; NR2A) and channel conductance.

7q21.11- GRM3 Metabotropic Group II metabotropic glutamate recep- [Harrison et al., 2008] 12 glutamate receptor (along with mGlu2), acting primar- tor 3 (mGlu3) ily as inhibitory autoreceptors.

1p21.3 MIR137 MicroRNA 137 Non-protein-coding gene. A microRNA, [Pasquinelli, 2012] which regulates other genes by binding to the 3’ untranslated region of their transcripts.

8p12 NRG1 Neuregulin 1 Growth factor, involved in many as- [Mei and Nave, 2014] pects of nervous system development and plasticity. Not GWAS signiﬁcant.

17p13.3 SRR Serine racemase Enzyme which synthesises D-serine from [Balu et al., 2013] L-serine.

18q21.2 TCF4 Transcription fac- Basic helix-loop-helix transcription fac- [Forrest et al., 2014] tor 4 tor. Haploinsuﬃciency causes Pitt- Hopkins syndrome.

2q32.1 ZNF804A Zinc ﬁnger pro- Putative transcription factor. [Hess and Glatt, tein 804A 2014]

28 Up to 30% of patients with schizophrenia show no or only partial response to treatment with at least two different antipsychotic medications [Con- ley and Kelly, 2001]. Identified predictive factors for treatment resistance include younger age, symptom severeness, previous antidepressant medica- tion, suicide attempt and affective symyptoms [Wimberley et al., 2016]. In those patients, the atypical antipsychotic clozapine was unequivocally confirmed to be the single most effective treatment choice, providing additional relief from some negative symptoms compared to other antipsychotics [Naber and Lambert, 2009, Kumar et al., 2017].

1.6 Clozapine

Clozapine (CLZ) is a tetracyclic compound with outstanding clinical eﬀec- tiveness in the treatment of psychotic symptoms even in patients resistant to high doses of typical neuroleptics [Attard and Taylor, 2012,Kumar et al., 2017].

Figure 1.18: Clozapine

Clozapine was the ﬁrst agent to challenge the prevailing dogma of potent dopamine D2 antagonism as a premise for antipsychotic eﬃcacy in the 1970s [Stille et al., 1971]. Its mechanism of action remains elusive, mak-

29 ing its study a central opportunity for the identiﬁcation of potentially novel targets for antipsychotic pharmacotherapy.

While clozapine shows superior antipsychotic efficacy, has effects on negative symptoms and evokes no extrapyramidal side effects opposed to classical antipsychotics, it remains a second or third line treatment. The incidence of drug induced agranulocytosis poses a potentially fatal hazard of clozapine treatment, substantially limiting its application [Wahlbeck et al., 1999]. While novel and effective genetic screenings for individual risk prediction were developed [Girardin et al., 2018], mandatory blood monitoring narrows the drug’s potential from an economic and patient compliance perspective. Clozapine treatment is strongly associated with weight gain through yet unknown pathways. A potential correlation between genetic variations in the GABRA2 gene and the risk of weight gain was recently identified [Zai et al., 2015]. With an incidence of 1.3%, clozapine treatment greatly increases the risk of generalized tonic-clonic seizures in a dose-dependant way [Pacia and Devinsky, 1994]. Additionally, risk for potentially fatal cardiomyopathy and myocarditis is markedly increased by CLZ treatment [Merrill et al., 2005]. Premature cardiovascular death presents a principal cause of morbidity in schizophrenic populations [Skalicky, 2017].

While long-term treatment with clozapine as well as haloperidol were found to upregulate GAT1 and GAD67 in rats [Zink et al., 2004a, Zink et al., 2004b], the increase of muscimol binding, reﬂecting GABAA receptor GABA binding sites in the infralimbic cortex remains a unique feature of clozapine [Zink et al., 2004b]. Korpi et al. proposed an antagonistic eﬀect of clozapine on GABAA receptors based on radioligand studies [Korpi et al.,

1995]. Assemblies containing α6 subunits were mainly unaﬀected by CLZ with the exception of the β1 containing assembly α6β1γ2. The widespread

α1β2γ2 assembly was inhibited by CLZ and eﬀects were not altered by application of ﬂumazenil, a compound with benzodiazepine-antagonistic prop-

30 erties [Korpi et al., 1995]. Additional in-vivo experiments further confirmed the GABAAR inhibiting effects of clozapine as well as one of its meabo- lites, N-desmethylclozapine, at high micromolar concentrations [Wong et al., 1996]. Another distinguishing property of CLZ is its effect on the noradrenergic system. It was proposed that α-2 receptor antagonism [Ashby and Wang, 1996], as well as the ability to block reuptake of norepinephrine following synaptic release [Khokhar et al., 2015] might take an important part in its unique clinical profile [Khokhar et al., 2018].

31 2. Materials and Methods

2.1 Two-Electrode Voltage Clamp

Two-electrode voltage clamp (TEVC) is a conventional electrophysiological technique suited for current measurements in large cells. Two electrodes are placed intracellularly. One electrode is used to inject current into the cell and adjust the membrane potential to desired values (voltage clamp). The other electrode is used to record changes in voltage resulting from the cell’s ion channel behaviour under a desired experimental setting.

2.1.1 Xaenopus laevis oocytes

Xenopus laevis oocytes present an established choice for the expression and study of plasma membrane proteins. Due to their physiological role, oocytes are rich in substrates and organelles, making them suitable expression systems. Additionally, Xenopus laevis oocytes express a relatively small number of ”idiogenic” ion channels, making them ideal for the study of recombinantly expressed channels.

2.1.2 Preparation of oocytes

The methods for isolation, culturing, injection and defolliculation of oocytes were identical with those described by E. Sigel [Sigel et al., 1990].

32 Stage 5-6 oocytes were obtained from the ovary lobe of previously anes- thesized and decapitated mature female Xenopus laevis (Nasco, Fort Atkin- son, WI, USA).

For separation from the sorrounding follicle layer, previously isolated oocytes were incubated in a solution of 1mg/ml collagenase (type IA, Sigma, MO, USA) in xenopus ringer (XR) composed of 90mM NaCl, 1mM MgCl2, 1mM KCl, 1mM CaCl2 and 5mM HEPES-NaOH (pH 7.4) for 60-90 min- utes. Removal of follicular layers after enzymatic digestion was achieved by pipetting cells through sterile pasteur pipettes. For storage periods a solution of 100 U/ml penicillin and 100µg/ml streptomycin in XR, referred to as XR+, was used.

Figure 2.1: Defolliculation process - modiﬁed from [Sigel and Minier, 2005]

After 24 hours of resting at 18◦C the cells were transferred to XR medium for injection of mRNA. Injection pipettes were pulled from glass capillaries using a P-97 puller (Sutter Instrument, California, USA) und subsequently polished under a Micro Forge MF830 microscope (NARISHIGE SCIEN- TIFIC, Tokyo, Japan). Between 2.5 ng and 4 ng of mRNA were injected into each cell using a Drummond Nanoject 2 injection device (Drummond Scientiﬁc Company, PA, USA).

33 For α β γ receptors, a subunit mRNA ratio of 1:1:5 was used, following the approach of Z. Varagic [Varagic et al., 2013], as described previously by E. Sigel and X. Li [Sigel et al., 1990, Li et al., 2003]. During incubation, cells were regularly checked to ensure prompt removal of dead or disintegrating cells from the bath. XR solutions were replaced daily.

2.1.3 Electrophysiological recordings

After incubation for at least 24 hours depending on subunit composition, oocytes were placed on a nylon-grid in a bath of XR solution. The oocytes were impaled with two glass microelectrodes (2-3 Mohm), which were filled with 2 M KCl. A constant flow of XR (6 mL/min) was then applied that could be switched to XR containing GABA and/or clozapine. The perfusion system is based on a gravity flow mechanism, aiming at constantly washing the cell while keeping mechanical stress at a minimum. Two valves provide the switches between delivery of washing solution and drug-containing solution to the oocyte. An Ismatec Reglo Digital I30 peristaltic pump (Cole-Parmer GmbH, Wertheim, Germany) removes excess solution from the oocyte bath at a constant rate. To test for modulation of GABA induced currents by clozapine, measurements were performed in presence of a GABA concentration that triggers 3-5 percent of the respective maximum GABA-elicited current of the individual oocyte (EC3-5). Determination of EC3-5 was performed via measurement of a full GABA dose response curve. The current measured during application of 1mM GABA was referenced as the maximum current. Clozapine was pre-dissolved in dimethyl sulfoxide (DMSO) to 30mM stock solutions and diluted in escalating concentrations in previously determined GABA EC3-5 XR.

34 Figure 2.2: GABA EC3-5 determination sequence

Between two applications, oocytes were washed with XR for up to 15 min to ensure full recovery of sensitivity. Before each clozapine sequence, two control measurements using GABA EC3-5 were performed. All recordings were performed at room temperature at a holding potential of -60 mV using a Warner OC-725C TEV (Warner Instrument, Hamden, CT, USA) or a Dagan TEV-200A TEV Oocyte clamp (Dagan Corporation, Mineapolis, MN, USA). Data was digitized, recorded and measured using a Digidata 1322A data acquisition system (Axon Instruments, Union City, CA, USA). Data was analyzed using Prism 6 (GraphPad, La Jolla, CA, USA). After elimination of outliers (CI95%) statistical significance was tested with paired t-test and 95% confidence interval. Oocytes expressing assemblies with the known high-affinity benzodiazepine binding site at the α+/γ2- interface were tested by application of diazepam at GABA EC3-5 to confirm receptor configuration.

35 3. Results

In preliminary screenings we applied 100µmol CLZ at different GABA concentrations, observing a maximum inhibition of GABA elicted currents at an EC3-5 of GABA. The observations are suggestive of a right shift of the GABA dose-response curve. Additionally, we gathered hints for a reduction of the maximum evoked GABA current at 1 µMol GABA. These preliminary findings are compatible with an allosteric (noncompetitive), negative modulatory mechanism of action. On this basis, further measurements were performed at an EC3-5 of GABA, in order to best portray CLZs effects.

Inhibition of GABA currents by CLZ in α1β1,2,3γ2 receptors was already demonstrated in earlier experiments [Korpi et al., 1995]. We successfully re- produced the described inhibitory eﬀects on α1β2γ2 receptors at micromolar concentrations of CLZ. In order to aid the understanding of potential mechanisms of action and binding sites, we studied CLZs eﬀects on four additional assemblies (α1β2,

α2β3γ2, α2β3γ1 and α2β3) with previously uncertain sensitivity. We focused especially on α2 containing assemblies due to their possible role as targets for drugs effective in treating some of schizophrenias symptoms [Charych et al., 2009]. A summary of the effects on all five measured receptor subtypes is given in Section 3.6.

36 3.1 α1β2

In α1β2 receptors, signiﬁcant inhibition of GABA elicted currents was observed at CLZ concentrations of 10 µmol, 30 µmol and 100 µmol. The mean inhibition reaches a maximum of 59% at 100 µmol CLZ concentration.

Figure 3.1: The range of observed currents at each CLZ concentration is represented by dark blue bars. After elimination of outliners (Q=1%), interpolation of a four parameter logistic regression curve was conducted. A 95% coinﬁdence interval (CI) of the curve is represented by the dotted line.

Table 3.1: α1β2