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Functional Expression and Characterization of Histamine-gated Chloride Channels Arunesh Saras Ph.D. Dissertation To be presented by permission of the department of Cellphysiology of the Ruhr-University, Bochum & International Graduate School of Neuroscience 2005

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  • Functional Expression and Characterization of Histamine-gated Chloride Channels Arunesh Saras Ph.D. Dissertation To be presented by permission of the department of Cellphysiology of the Ruhr-University, Bochum

    & International Graduate School of Neuroscience

    2005

  • Contents

    i

    Contents Chapter 1: Introduction

    1.1 GABA-receptors and the GABAergic system

    1.1.1 The inhibitory -aminobutyric acid system 1

    - a general overview

    1.1.2 GABA-receptors: GABAB and GABAC 1

    1.1.3 General properties of GABAA receptors 2

    1.1.4 Types of heteromultimeric GABAA receptors 4

    and their location and properties

    1.1.5 Homomultimeric GABAA receptors 5

    1.1.6 Trafficking of GABA-receptors and interacting proteins 6

    1.1.7 Potentiation and modulation of GABAA receptors 8

    1.1.7.1 Modulation of GABAA receptors by Propofol 8

    1.1.7.2 Modulation of GABAA receptors by further 13

    chemicals

    1.1.8 Function of distinct GABAA subunits in vivo 14

    investigated by knockout mice

    1.2 Histamine-receptors and the histaminergic system 17

    1.2.1 Histamine in the nervous system 17

    1.2.2 Metabotropic histamine receptors 18

    1.2.3 Interaction of histamine antagonists with GABAA receptors 22

    1.2.4 Ionotropic histamine receptors and direct modulatory 22

    action of histamine to ion channels

    1.2.5 Histamine functions and knockout mice 24

    1.2.6 Diseases where histamine is involved 25

    1.2.7 Aims of the work 27

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    Chapter 2: Materials 28

    2.1 Chemicals and enzymes 28

    2.2 Drugs used for pharmacological characterizations 29

    2.3 Primers 31

    2.4 Standards for DNA 32

    2.5 Consumption materials 32

    2.6 Kits 33

    2.7 RNase free materials and chemicals 33

    2.8 Frequently used buffer 34

    2.9 Bacterial strains 37

    2.10 Plasmid vectors 37

    2.11 Softwares 38

    Chapter 3: Methods 39

    3.1 Characterizing, isolating and concentrating nucleic acids 39

    3.1.1 Determination of concentrations of nucleic acids 39

    3.1.2 Gel electrophoresis 39

    3.1.3 Phenol: chloroform extraction of nucleic acids 39

    3.1.4 Ethanol precipitation of nucleic acids 40

    3.1.5 QIAquick PCR-Purification Kit 41

    3.1.6 RNA Extraction 42

    3.1.7 Quick preparation of plasmid DNA 42

    3.1.8 Maxi preparation of plasmid DNA using the 43

    QIAGEN Plasmid Maxi Kit

    3.2 PCR Methods 43

    3.2.1 Reverse transcription 43

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    3.2.3 PCR-based generation of chimeric cDNAs and 45 site-directed mutagenesis

    3.2.4 Phosphorylation op PCR primers 46

    3.3 Cloning of DNA 46

    3.3.1 Restriction 46

    3.3.2 Dephosphorylation 46

    3.3.3 Polishing of DNA using T4 DNA polymerase and T4 PNK 47

    3.3.4 Fill in reaction 47

    3.3.5 Ligation of DNA 48

    3.3.6 Culturing of bacteria 48

    3.3.7 Transformation of plasmid DNA 48

    3.3.8 Sequencing of DNA 48

    3.4 RNA techniques 48

    3.4.1 In vitro transcription 49

    3.5 Functional expression of LGICs in Xenopus laevis 50

    3.5.1 Surgery 50

    3.5.2 Oocyte preparation and injection of cRNA 50

    3.5.3 Electrophysiological recording using two-electrode 51

    voltage clamp

    3.6 Functional Expression of LGICs in HEK 293 cells 52

    3.6.1 Culture of HEK 293 cells and transfection 52

    3.6.2 Patch clamp investigation of GABA receptors 53

    expressed in HEK 293 cells

    Chapter 4: Results I 55

    4.1 Bioinformatical search for histamine-gated channels 55

    4.2 Construction of expression vectors for GABAA receptors 56

    4.3 Establishing functional expression GABAA receptors in 61

    Xenopus oocytes

    4.4 Direct effects of histamine on heteromultimeric GABAA receptors 65

    4.5 Modulation of heteromultimeric GABAA receptors by histamine 65

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    4.5.1 Potentiation of 11 GABAA receptors by histamine 65

    4.6.1 Potentiation of 11 GABAA receptors by histidine 69

    4.6.2 Characterization of histidine potentiation 70

    4.6.3 Dependence of average histamine and histidine 72

    potentiation on GABA concentration

    4.6.4 Histidine does not alter the I/V curve and retains 73

    selectivity for the permeability of the channel

    4.7 Potentiation of GABAA receptors in HEK 293 cells 74

    4.8 Dependence of histamine potentiation on GABAA receptor 76

    subunit combinations

    4.9 Homomultimeric channels of 1 subunit and the effect of 81

    histamine and histidine

    4.9.1 Homomultimeric channels of 3 subunit and effect of histamine 84

    4.10 Molecular cloning of 1 subunit of GABAC receptors 84

    4.10.1 GABAC receptors: No potentiation by histamine and histidine 86

    4.11 Possible mechanisms of the histamine action 87

    4.11.1 Histamine binding site is different from pentobarbital 87

    binding-site on 3 subunit of GABAA receptors

    4.11.2 Histamine binding site is similar to propofol binding- 88

    site on 3 subunit of GABAA receptors

    Experiments on homomultimeric 3 subunit

    4.11.3 Histamine binding site is similar to propofol binding 90

    Site on 3 subunit of GABAA receptors

    - Experiments on heteromultimeric 11 receptors

    4.11.4 Effect of histamine on EC50 of GABA on GABAA receptors 91

    4.11.5 Molecular cloning of point mutation in 1 subunits 96

    4.11.6 1(M286W) mutation completely abolishes potentitaion 96

    mediated by histamine

    4.11.7 Histamine and propofol have similar binding sites 98

    4.11.8 1 (M286W) mutation completely abolishes potentitaion 101

    mediated by histidine

    4.11.9 Sequence alignment with GABAC receptors depicts that 101

    histamine has similar binding site to propofol

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    4.11.10 1 (M286W) mutation does not interfere with the 102

    potentiaion mediated by other modulators

    Chapter 5: Results II 106

    5. Characterization of homomultimeric 3 channels 106

    5.1 Homomultimeric 3 receptors behave like histamine-gated ion channels 106

    5.2 Histamine gated homomultimeric 3 receptors behave like typical 106

    ligand-gated chloride channels

    5.3 Relative comparison of various agonist of histamine with GABA 110

    - relative agonists efficacy compared to GABA

    5.4 Pharmacological characterization of histamine-gated homomultimeric 112

    3 receptors

    5.4.1 Thioperamide acts as a competitive blocker for 113

    histamine-evoked current

    5.5 Comparison of L- and D-histidine action on 3 homomultimeric receptors 116

    5.6 Inhibition of Propofol-induced current by thioperamide on 118

    homomultimeric 3 receptors

    Chapter 6: Results III 121

    6. Characterization of homomulitmeric 2 channels 121

    6.1 Homomultimeric 2 receptors behave like histamine-gated ion channels 121

    6.2 Histamine gated homomultimeric 2 receptors behave like typical ligand- 122

    gated chloride channels.

    6.3 Relative comparison of various agonists of histamine with GABA 122

    6.4 Pharmacological characterization of histamine-gated homomultimeric 122

    2 receptors

    6.5 Relative comparison of homomultimeric 2 and 3 homomultimeric 126

    receptors

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    Chapter 7: Results IV 132

    7. Characterization of homomultimeric subunit 132

    7.1 Characterization of subunit containing receptors 132

    7.2 contianing receptors behave like histamine-gated ion channels 133

    7.3 Pharmacology of subunit 135

    7.4 Expression of 11, 1 and in HEK 293 cells 136

    7.5 Molecular cloning of GFP-tagged subunits of GABAA receptors 136

    7.5.1 Molecular cloning of GFP-tagged 1 and 1 subunits of 137

    GABAA receptors

    7.5.2 Molecular cloning of GFP-tagged subunit of 137

    GABAA receptor

    7.6 Expression of 1, 1 and in HEK 293 cells 142

    Chapter 8: Results V 145

    8.1 Properties of 122 receptors and direct activation by histamine 145

    8.2 Homomultimeric 2 receptors: Activation by histamine and its 147

    metabolites

    Chapter 9: Discussion 150

    Chapter 10: Summary 167

    Chapter 11: References 169

    Appendix: 189

    Appendix I 189

    Appendix II 190

    Acknowledgements 191

    Curriculum Vitae 192

  • Chapter 1 Introduction

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    1. INTRODUCTION

    1.1: GABA-receptors and the GABAergic system

    1.1.1: The inhibitory -aminobutyric acid system - a general overview

    Gammaaminobutyric acid (GABA) is the major inhibitory neurotransmitter in the

    mammalian central nervous system. It regulates many physiological functions and

    emotional and cognitive behaviors through neurosynaptic contacts widespread in the

    brain (Costa, 1982). In the mammalian brain the GABA is synthesized primarily from

    glutamate in a reaction which is catalyzed by two glutamic acid decarboxylase (GAD)

    enzymes, GAD65 and GAD67 (Bloom and Iversen, 1971). In the synaptic vesicle GABA

    is loaded by a vesicular neurotransporter (VGAT) (Fon and Edwards, 2001) and it is

    liberated into nerve terminal by calcium dependent exocytosis. However, no-vesicular

    GABA secretion is being described and might play a role during development (Attwell et

    al., 1993; Taylor and Gordon-Weeks, 1991). The effect of GABA can be mediated by

    ionotropic or metabotropic receptors, which are localized post - or presynaptically. The

    termination of GABA activation can happen either by its reuptake into the nerve

    terminals or surrounding glial cells by a class of plasma membrane GABA transporters

    (GATs) (Cherubini and Conti, 2001). Thereafter, GABA is metabolized by a

    transamination reaction that is catalyzed by GABA transaminase (GABA-T). GABA acts

    on 3 types of receptors which are phylogenitically conserved across different species:

    GABAA, GABAB and GABAC receptors (Friedl et al., 1988).

    1.1.2: GABA-receptors: GABAB and GABAC

    GABAB receptors are bicuculline insensitive, chloride independent, metabotropic

    receptors (Hill and Bowery, 1981; Bowery et al., 1980; Nicoll, 1988) and belong to the

    superfamily of G-protein coupled receptors. GABAB receptors were shown to mediate

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    presynaptic inhibition on some nerve endings and postsynaptic inhibition on some cell

    bodies or dendrites. GABAB receptors exist as GABAB1a / GABAB2 and GABAB1b /

    GABAB2 and are associated with Gproteins. They have seven transmembrane domains.

    GABAB receptors are localized both pre- and postsynaptically and they use different

    mechanisms at these locations to regulate cell excitability. Presynaptic inhibition occurs

    through a GABAB receptor mediated reduction in calcium current at the nerve terminal

    and a subsequent reduction in transmitter release, whereas postsynaptic inhibition occurs

    by GABAB receptor mediated activation of potassium currents that hyperpolarize the

    neuron (Connors et al., 1988).

    Like GABAA receptors, GABAC receptors are ligand-gated ion channel receptors (Sigel,

    1995; Johnston, 1996; Enz and Cutting, 1998). This receptor is a chloride-selective ion

    channel, but is insensitive to the GABAA receptor antagonist bicuculline (Bormann and

    Feigenspan, 1995). GABAC receptors are believed to be homo - or heteropentameric

    proteins that are composed of a single or multiple subunits. They are also different from

    GABAB receptors being insensitive to baclofen but are responsive to cis-4-aminocrotonic

    acid, a structural analogue of GABA. GABAC receptors can be considered as

    pharmacological variants of GABAA receptors (Mehta and Ticku, 1999; Bormann, 2000).

    1.1.3: General properties of GABAA receptors

    The GABAA receptors are members of the ligand-gated ion channel superfamily, which

    also includes nicotinic acetylcholine, glycine and serotonin (5-HT3) receptors. GABAA

    receptors are the primary mediators of GABA-induced rapid inhibitory neurotransmission

    (Sieghart, 1995) and are believed to be heteropentameric proteins that are constructed

    from subunits derived from several related genes or gene families (Macdonald and Olsen,

    1994). At present, six subunits, three subunits, three subunits, one subunit, one

    subunit, one subunit and one subunit have been identified in mammals (Macdonald

    and Olsen, 1994; Schofield et al., 1987; Mehta and Ticku, 1999). All the subunits are

    related to each other and have molecular weights of about 50 kD. These various subunits

    provide enormous subunit combinations but only certain subunit combinations are

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    preferred (McKernan and Whiting, 1996). Native receptors contain at least one , one

    and one subunit. The , , and subunits able to substitute for the subunit

    (McKernan and Whiting, 1996). GABAA receptors are integral membrane proteins,

    which are formed by assembly of five homologous subunits around a central ion channel

    (Chang et al., 1996). Each subunit has a large extracellular N-terminal domain and a C-

    terminal domain containing four transmembrane segments, designated M1-M4, and

    connected by relatively short loops. The extracellular N-terminal domains are believed to

    form the agonist binding sites, whereas the transmembrane domains form the channel;

    with the five M2 domains being the primary lining of the ion-conducting pore of the

    receptor (Xu and Akabas, 1996). The M2 domain is thought to be a key channel-lining

    component, which determines channel properties such as conductance, rectification, and

    desensitization. Determined by the pore-forming M2-region, GABAA receptors carry

    primarily chloride ions, however other anions, such as bicarbonate (HCO3-) can also

    permeate the channel pore, although less efficiently (Kaila, 1994; Bormann et al., 1987;

    Moss and Smart 2001).

    Fig. 1.1: Structure of GABAA receptors. Proposed structure of a ligand gated ion channel. A receptor subunit contains four hydrophobic transmembrane (TM) domains. TM2 is believed to form the lining of the ion channel. The large amino terminal domain is located extracellularly and believed to incorporate neurotransmitter and some modulators binding sites. The intracellular domain in between TM3 and TM4 comprises ~ 10 % of the mass of each subunit. This domain is the most divergent part of individual receptor subunits and contains numerous consensus sites for the action of both serine/threonine and lysine protein kinases. Adapted from (Moss and Smart, 2001).

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    1.1.4: Types of heteromultimeric GABAA receptors and their location and

    properties

    Although molecular biology revealed seven types of homologous GABAA subunit types

    by now, the subunit composition and the arrangement of subunits within a functional

    GABAA receptor in the brain remains unknown in detail. Of the many subunit

    combinations that are theoretically possible, only a few dozen have been shown to exist,

    reflecting the differential distribution of subunit types among brain regions (Wisden et

    al., 1992; Fritschy and Mohler, 1995; Pirker et al., 2000). The most abundantly

    expressed receptor subtype in the brain is formed from 1, 2 and 2 subunits (Sieghart

    and Sperk, 2002; McKernan and Whiting, 1996; Whiting, 2003). The likely

    stoichiometry is two , two and one subunit (Tretter et al., 1997; Farrar et al., 1999),

    with the subunits arranged around the ion channel pore in the sequence ----,

    (Baumann et al., 2002). Other common assemblies also contain , and 2 subunits (for

    example, 232, 332, 4x2, 532 and 6x2), whereas receptors in which the 2

    subunit is replaced by 1, 3, or are less abundant. Further variability arises from the fact

    that individual pentamers might contain two different or two different subunit

    isoforms (Sieghart and Sperk, 2002). In some cases, the subunit can be replaced by , ,

    or subunit, and the and subunits might also be capable of co-assembling with ,

    and subunits to form receptors that contain subunits from four families (Neelands and

    Macdonald, 1999; Bonnert et al., 1999). This molecular heterogeneity has important

    functional consequences for GABAA receptor subtypes: subunit composition dictates not

    only the properties of the receptors, but also their cell surface distribution and dynamic

    regulation (Luscher and Keller, 2004; Sieghart and Sperk, 2002; Hevers and Luddens,

    1998).

    A combination of several methods allowed more precise subcellular localization of

    GABAA receptors, and enrichment of the 1, 2, 3, 6, 2, 3 and 2 subunits within the

    postsynaptic membrane of GABAergic synapses. Each of these receptor subunits was

    also found in extrasynaptic plasma membranes, and no GABAA receptor subunit type has

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    yet been found to have an exclusively synaptic location. Even in the case of 122

    GABAA receptors, which are highly enriched in synapses, more receptors are found

    outside than inside synaptic junctions. Some GABAA receptors do not seem to

    accumulate at synaptic junctions; for example, the subunit was shown to be present

    exclusively in the extra - synaptic somatic and dendritic membranes of cerebellar granule

    cells (Nusser et al., 1995) and at extra-synaptic and peri-synaptic locations in

    hippocampal dentate gyrus granule cells (Wei et al., 2003). The lack of a subunit is

    probably responsible for subunit failure to be incorporated at the synapse and subunit

    containing receptors seems to be purely extra - synaptic. In general, receptors containing

    a 2 subunit in association with 1, 2, 3 subunits are the predominant receptor subtypes

    that mediate synaptic inhibition and receptors that contain 4 or 6 subunits in

    combination with subunits are predominantly or exclusively extra synaptic.

    A vital property of a ligand-gated ion channel is its sensitivity to endogenous agonists.

    For recombinant receptors that contain , and subunit, sensitivity to GABA is most

    strongly affected by the type of subunit that is present, with 3 subunits conferring the

    highest and 6 subunits the lowest EC50 values (Knoflach et al., 1996; Fisher and

    Macdonald, 1997; Bohme et al., 2004; 2004; Minier and Sigel, 2004). The absolute

    EC50 values for specific subunit combinations reported by different groups is

    considerably variable, but, in studies in which subunits have been compared, the rank

    order was shown to be 6 < 1 < 2 < 4 < 5 < 3, Bohme et al., 2004). Replacing the

    2 subunit in 432 assemblies with a subunit decreases the EC50 for GABA (Brown et

    al. 2002). Overall 632 or 63 combinations have the lowest EC50s for GABA (~0.3

    0.7 M), whereas for 132 or 232 subtypes they are an order of magnitude higher

    (~614 M).

    1.1.5: Homomultimeric GABAA receptors

    It is reported that some GABAA receptor subunits indeed form homomultimeric channels.

    Among these subunits, the subunit is thought to be a key component to assemble

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    heteromultimeric functional ion channels, to play a central role in determining the

    subcellular locations of GABAA receptors (Connolly et al., 1996) and to bear binding

    sites for agonists (Sigel et al., 1990; Amin and Weiss, 1993) and some clinically

    important drugs such as general anesthetics (Cestari et al., 1996; Hill-Venning et al.,

    1997). The subunits are found to be capable of forming homomultimeric functional

    channels when expressed in Xenopus oocytes or mammalian cells (Sigel et al., 1989;

    Krishek et al., 1996). Channels composed of 1 subunits are constitutively active and

    show spontaneous currents whereas GABAA receptors that contain 3 subunits are

    inactive in the absence of GABA but they also form homomulitmeric channels, in which

    the GABA current can be potentiated by pentobarbital and propofol. It was shown

    (Martinez-Torres and Miledi, 2004) that the human 2 subunit could also form

    homomultimeric channels with an EC50 of 300 M. The 2 receptors were blocked by

    bicuculline and were potentiated by pentobarbital and flunitrazepam. The other possible

    homomultimeric receptors are suspected to be retained in the endoplasmic reticulum by

    interactions with the Ig-binding protein BiP or calnexin and are then rapidly degraded

    (Bollan et al., 2003; Gorrie et al., 1997).

    1.1.6: Trafficking of GABA-receptors and interacting proteins

    It is documented that GABAA receptors can be inserted and removed rapidly at synapses

    (Kittler and Moss, 2003). This process is important in the synaptic inhibition and causes

    the enhancement in the amplitude of miniature postsynaptic currents (mIPSC) (Wan et

    al., 1997). Insulin induces the rapid insertion of GABAA receptors in to the synaptic

    membrane by phosphorylating subunits through Phosphoinositide-3 Kinase (PI3K)

    (Wang et al., 2003). Conversely, removal of the receptors occurs by the activity of brain

    derived neurotrophic factors (NFS) leading to suppression of mIPSC (Jovanovic et al.,

    2004). Like glutamate receptors, there are both relatively immobile and highly mobile

    GABAA receptors on the surface of neurons (Velazquez et al., 1989), with certain

    subunits (for example, 1 and 6) being responsible for anchoring at the surface (Peran et

    al., 2004). Several proteins have been identified that bind directly to GABAA to regulate

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    their trafficking and to determine the role of phosphorylation in this process (Moss and

    Smart, 2001).

    Fig. 1.2: Dynamic control of GABAA receptor expression at the cell surface. GABAA receptors composed of , and subunits are clustered at synaptic sites by both gephyrin-dependent and independent mechanisms, the details of which remain unresolved. GABAA receptors are intimately associated with both protein kinsaes C II (PKC-II) and RACK 1 (receptor associated C kinase), which together mediate the phophorylation of receptor subunits, facilitating functional modulation of GABAA receptors. In addition, GABAA receptors at synaptic sites undergo constitutive dynamin-dependent, clathrin-medited exocytosis. This process is mediated by the interaction of receptor subunits with and adaptins, proteins that are essential for the recruitment of cargo in to cathrin-coated pits. Internalized receptor enters the endosomal system, where they can be returned to the cell surface. Interaction with the GABA receptor 2 subunit might facilitate membrane interaction of GABAA receptors. This protein specifically localized into intracellular membrane including the Golgi net, Moreover GABARAP can bind NSF (N-ethylmaleimide sensitive factor), a key protein in membrane fusion that is critical for intracellular transport. Adapted from (Moss and Smart, 2001).

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    Direct binding partners include GABA-receptor-associated protein (GABARAP) (Wang

    and Olsen, 2000; Wang et al., 1999), receptor for activated C-kinase (RACK1) (Brandon

    et al., 1999), Src and AP2 (Mochly-Rosen and Gordon, 1998; Chang et al., 1998;

    Yarwood et al., 1999). The GABA receptors are retained in the Golgi complex by

    GABARAP protein (Wang et al., 1999) and their exit from this compartment could

    involve interactions between GABARAP and N-ethyl maleimide sensitive factor (NSF)

    and / or catalytically inactive phospholipase C (PLC)-related protein. Once inserted at

    synapses, GABAA receptors are stabilized by their interaction with gephyrin and other

    clustering molecules (Kneussel et al., 2001). Endocytosis of GABAA receptors might also

    involve an interaction with (ubiquitin related protein), Plic-1, which could protect them

    from degradation (Bedford et al., 2001).

    1.1.7: Potentiation and modulation of GABAA receptors

    The GABAA receptors are modulated by various chemical agents like benzodiazepines

    (Sigel, 2002; Boileau and Czajkowski, 1999), neurosteroids (Rick et al., 1998),

    barbiturates (Olsen et al., 1986), anesthetics (Krasowski et al., 1998) and alcohol (Mihic

    et al., 1997). In total, GABAA receptors incorporate more than ten distinct binding sites

    which have made this receptor a well recognized target for drug development (Korpi,

    1994). In this study, it turned out that the modulatory site for propofol was the most

    important one; therefore it is described in greater detail.

    1.1.7.1: Modulation of GABAA receptors by Propofol

    Propofol belongs to the class of general anesthetics enhancing GABAA-receptor function.

    General anesthetic administration induces a state characterized by loss of consciousness,

    amnesia, analgesia and immobility (Yamakura et al., 2001). At the level of ion channels,

    intravenous anesthetic effects on GABAA receptors are concentration dependent. At low

    concentrations, GABA-active anesthetic potentiate submaximal GABA-induced currents.

    At higher concentration, they directly open channels in the absence of GABA

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    (Yamakura et al., 2001; Belleli et al., 1999). At even higher concentrations, some

    anesthetics inhibit currents.

    Fig. 1.3: Modulation of GABAA receptors by various chemicals. Several classes of CNS depressant drugs like benzodiazepines, barbiturates, neurosteroids, anesthetic, alcohol and propofol have been suggested to modulate GABAA receptors. In particular, the anxiolytic effects of benzodiazepines are shown to result from potentiation of GABA action on GABAA receptors. Barbiturates and related sedatives also enhance GABAA receptor-mediated inhibition, and their pharmacological spectrum overlaps with that of the benzodiazepines and related substances. The two classes of drugs have a different mechanism of action at the molecular channel level; barbiturates prolong the lifetime of GABA currents, in addition to gating channels directly at high concentrations, whereas benzodiazepines increase the frequency of opening of GABA receptor channels and do not directly open channels in the absence of GABA. Long-chain alcohols have anesthetic activity, as does ethanol at high doses (greater than 100 mM), whereas the intoxicating effects at lower concentrations (10 to 100 mM) have been suggested to involve enhancement of GABAA receptors. GABAA receptor function is further modulated by neurosteroids (Progesterone etc.). The neuroactive steroids act principally by binding directly to membrane GABAA receptors and enhancing their function in a manner resembling the barbiturates. Propofol is an alkylphenol derivative (2, 6 - diisopropylphenol), which is a fast-acting, short-lived intravenous anesthetic. It has similar mechanism of action like benzodiazepines. Adapted from (Rudolph and Antkowiak, 2004).

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    Propofol is an alkylphenol derivative (2, 6 - diisopropylphenol), which is a fast-acting,

    short-lived intravenous anesthetic. The behavioral actions of propofol cover a large

    concentration range. High concentration produces sleep, sedation, hypnosis and

    immobility, whereas mild sedation and impairment of memory occurs at lower

    concentration (around 3 % of those needed to induce immobility) (Veselis et al., 2002;

    Smith et al., 1994). At sedative concentration, propofol reduces neuronal activity

    prominently in cortical networks. At higher, hypnotic concentration, subcortical

    structures, including the thalamus, midbrain reticular formation and possibly the

    hypothalamus, are also affected (Rudolph and Antkowiak, 2004). Interestingly, there is a

    linear relationship between the regional benzodiazepine binding site densities, consistent

    with a similar mechanism of action of propofol and benzodiazpines (Alkire and Haier,

    2001).

    During propofol-induced hypnosis, global cerebral blood flow and glucose metabolism

    seem to be significantly decreased, and some brain areas show a markedly higher degree

    of depression than others. These regions are localized in diverse cortical areas, and also in

    the thalamus and midbrain (Fiset et al., 1999; Alkire, 1998). Electroencephalography

    (EEG) (Alkire, 1998) has provided evidence that thalamic structures are inhibited at

    hypnotic propofol concentrations. In an elegant approach (Hofbauer et al., 2004) showed

    that at mildly sedating concentration, human subjects ratings of thermal pains were

    increased, and there was a corresponding increase in evoked activity in the thalamus and

    somatosensory cortex. When subjects lost consciousness, noxious stimuli evoked

    thalamic responses were abolished. Bonhomme used a similar experimental design,

    tactile stimuli were applied during sedative and hypnotic propofol

    concentration (Bonhomme et al., 2001). With hypnotic concentrations of propofol,

    thalamic and cortical responses ceased. Magoun and Moruzzi found that several nuclei in

    the midbrain reticular formation are involved in arousal, wakefulness and sleep and these

    structures are plausible targets for general anesthetic to produce some of their sedative

    and hypnotic effects (Moruzzi and Magon, 1949). General anesthesia and sleep share

    some common features like depression of sensory input and motor output and similar

    EEG patterns. Moreover, similar to sleep a recovery process takes place in anesthesia

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    (Tung et al., 2004). So, hypothalamic networks that are involved in sleep regulation

    might have a key role in mediating anesthetic-induced hypnosis. The hypnotic effects of

    several anesthetic applied to TM nucleus-hypothalamic region involved in regulation of

    sleep and wakefulness, are consistent with such a mechanism (Nelson et al., 2002).

    Fig. 1.4: Propofol anesthesia in humans. (a) Correlation between propofol and plasma concentration and anesthetic depth. Symbols indicate values reported in different studies to cause light and moderate sedation, hypnosis and immobility. Horizontal bars represent mean values. (b) Sites in the CNS that are thought to be involved in the sedative, hypnotic and immobilizing actions of propofol. The concentration-dependent depression of CNS functions by propofol seems to be in accordance with the classical idea that phylogenitically older parts of the CNS are more resistant to anesthetic treatment than those that appeared later in evolution. Adapted from (Rudolph and Antkowiak, 2004).

    Neuroanatomical substrates that are relevant for the modulation of working memory by

    general anesthetic have been identified by functional imaging studies, in which human

    subject were asked to memorize words during administration of propofol (Veselis et al.,

    2002). Propofol causes similar concentration-dependent depression of regional cerebral

    blood flow (rCBF) and oxidative metabolism in the brain so it is reasonable to assume

    that propofol induced depression in rCBF is closely linked to a depression in neuronal

    activity (Kaisti et al., 2002).

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    The GABAA receptors have attracted considerable attention as a target for anesthetic

    agents. Using knock-in point mutations in mice, (Jurd et al., 2003) have provided

    definitive evidence that specific GABAA receptors are involved in the actions of propofol.

    Whereas sites on both and subunits are crucial for volatile anesthetic action (Mihic et

    al., 1997) for example 1-S270, 1-A291, 2-N265 and 2-M286, only the sites on

    subunits have been found to be relevant for the actions of the intravenous anesthetic

    propofol (Krasowski et al., 1998). Two groups have recently reported the introduction of

    point mutation in to subunits on the GABAA receptors. Jurd showed the generation and

    analysis of 3 (N265M) mice (Jurd et al., 2003). This point mutation abolished the

    modulatory and direct effect of etomidate and propofol in vitro, and subsequently

    reduced the modulatory actions of enflurane, whereas the modulatory actions of

    neuroactive steroid alphaxalone was preserved (Siegwart et al., 2002). The duration of

    the loss of the righting reflex in response to etomidate and propofol was reduced in 3 (N265M) mice compared with wild-type mice, indicating that the hypnotic activity is

    mediated in part by GABAA receptors that contain the 3 subunit and in part by other

    targets, possibly GABAA receptors that contain the 2 subunit. A point mutation in 1

    subunit (M286W) abolished potentiation of GABA by propofol but did not alter direct

    activation of the receptor by higher concentrations of propofol (Krasowski et al., 1998).

    This point mutation in M3 of the 1 subunit (M286W) eliminated GABA potentiation by

    1 M propofol. In fact, submaximal GABA currents at the 21 (M286W) mutant

    receptor were not enhanced by propofol at concentration up to 10 M. Cysteine

    substituted for these residues was used to determine whether propofol could protect it by

    sulfhydryl reactive reagents p-chloromercuribenzensulfonate (pCMBS) (Bali and

    Akabas, 2004). The pCMBS reaction rate with an engineered Cys depends on two major

    factors: first accessibility of the Cys to bulk solution and second reactivity of the Cys

    with sulfhydrylreagents. Accessibilty depends on stearic and electrostatic factors in the

    access pathway from bulk solution to the site of Cys. Their results showed that propofol

    protected the substituted Cys at 2 M286W by a stearic effect caused by the local

    presence of propofol and hence they concluded that this residue lies near the propofol-

    binding site. The other 2 subunit residue, 2N265C was not protected by propofol.

  • Chapter 1 Introduction

    13

    Methionine oocupies a volume 43 A greater than asparagine and is more hydrophobic.

    Thus, stearic bulk at position 265 can alter propofol binding, perhaps by inducing a

    conformational change at the propofol binding site equivalent 100 A away.

    Fig. 1.5: Proposed roles of GABAA-receptor subtypes and other target in propofol actions. Propofol acts on various targets including voltage-dependent Na+ channels to various subunits of GABAA receptors. Action of propofol on 2 and 3 subunits of GABAA receptors leads to sedation, hypnosis and immobility whereas its activity on glycine receptors, L-type Ca2+ channels is unclear. Adapted from (Rudolph and Antkowiak, 2004).

    1.1.7.2: Modulation of GABAA receptors by further chemicals

    Several classes of CNS depressant drugs had for some time been suggested to modulate

    GABAA receptors. In particular, the anxiolytic effects of benzodiazepines were shown to

    result from potentiation of GABA action on GABAA receptors (Costa et al., 1975). The

    classical benzodiazepines such as diazepam (Valium) have had a tremendous history in

    psychopharmacology primarily for clinical anxiety (Zorumski and Isenberg, 1991).

  • Chapter 1 Introduction

    14

    Other uses of benzodiazepines include sedation, muscle relaxation, and a significant

    utilization for treatment of panic (Biggio et al., 1995). Barbiturates and related sedatives also

    enhance GABAA receptor-mediated inhibition, and their pharmacological spectrum

    overlaps with that of the benzodiazepines and related substances. The selective actions of

    benzodiazepines not shown by barbiturates or vice versa arise from heterogeneity in

    GABA receptor sensitivity to the drugs, and corresponding heterogeneity in brain regions

    and functions. Some GABA-receptors are insensitive to benzodiazepines but not to

    barbiturates. In addition, the two classes of drugs have a different mechanism of action at

    the molecular channel level; barbiturates prolong the lifetime of GABA currents, in

    addition to gating channels directly at high concentrations, whereas benzodiazepines

    increase the frequency of opening of GABA receptor channels and do not directly open

    channels in the absence of GABA (Study and Barker, 1981). Alcohols are CNS

    depressants known to enhance GABAA receptor currents with a pharmacological spectrum

    of action overlapping those of the benzodiazepines and barbiturates. Long-chain alcohols

    have anesthetic activity, as does ethanol at high doses (greater than 100 mM), whereas the

    intoxicating effects at lower concentrations (10 to 100 mM) have been suggested to

    involve enhancement of GABAA receptors (Suzdak et al., 1986). GABAA receptor function

    is further modulated by neurosteroids. The neurosteroids are endogenous steroid hormone

    metabolites that have direct and rapid actions on cells not involving steroid hormone

    receptors or regulation of gene expression. Progesterone was shown to produce rapid

    sedative activity. Progesterone has anxiolytic and anticonvulsant activity; discontinuation

    after long-term administration leads to withdrawal signs that are clearly CNS mediated.

    The neuroactive steroids act principally by binding directly to membrane GABAA

    receptors and enhancing their function in a manner resembling the barbiturates (Lambert et

    al., 1995).

    1.1.8: Function of distinct GABAA subunits in vivo investigated by knockout

    mice

    Gene targeting and transgenic mice have demonstrated several important roles for GABA in

    the CNS. Knockouts of both GAD67 and GABAA receptor subunit 3 lead to early

  • Chapter 1 Introduction

    15

    neonatal lethality (Asada et al., 1997). GAD65 knockout mice show increased anxiety,

    increased sensitivity to benzodiazepines (Kash et al., 1999). Epilepsy results from knockout

    of GAD65, GABA 3, and GABA receptor subunit. Mice targeted for this subunit have

    a phenotype remarkably similar to Angelman syndrome, especially the epilepsy, but also

    including the cognitive, motor and sleep impairment (DeLorey et al., 1998). The 2 subunit

    knockout mice show early neonatal lethality (Gunther et al., 1995), without cleft palate,

    involving impaired clustering of GABAA receptors at synapses (Essrich et al., 1998).

    Because GABA receptors are important drug targets, some GABA receptors subunit

    knockout mice have impaired sensitivity to drugs, such as decreased response to

    benzodiazepines in 2 homozygous knockouts. Increased response to benzodiazepines is seen in 2 heterozygous knockouts or in 2L null mutants (Quinlan et al., 2000). Reduced

    sensitivity to anesthetics was seen in 3 but not 6 knockouts, and reduced sensitivity to

    neuroactive steroids is observed in the subunit knockout (Mihalek et al., 1999).

    Gene targeting in mice also has been employed to knock in a mutation of the 1 subunit

    H101N, which prevents benzodiazepine binding to GABA receptors containing this subunit

    (McKernan et al., 2000). The resulting animals have greatly impaired sensitivity to the

    sedative but not the anxiolytic actions of the benzodiazepines, whereas anticonvulsant

    activity is partially reduced. This finding indicates that the subtypes of GABA receptors

    containing the 1 subunit and the brain circuits in which they function are the substrates

    for benzodiazepine-stimulated sedation.

    1 subunit-containing GABAA receptors in forebrain contribute to the effect of inhaled

    anesthetics on conditioned fear. Knockout mice were 75 to 145 % less sensitive to the amnestic effects of the inhaled anesthetic isoflurane. These results indicate that 1-

    containing GABAA receptors in the hippocampus, amygdala, and / or cortex influence the

    amnestic effects of inhaled anesthetics (Sonner et al., 2005). Also 1 knockout mice show impaired dendritic spine maturation. There was a concomitant decreased density of

    mature mushroom-shaped spines, which became more pronounced in adults. In contrast,

    dendritic arborization was not altered in these mice (Heinen et al., 2003). 5 knockout

  • Chapter 1 Introduction

    16

    mice showed enhanced learning and memory and altered GABAergic synaptic transmission (Collinson et al., 2002). In the CA1 region of hippocampal brain slices from

    5 knockout mice, the amplitude of the IPSCs was decreased, and paired-pulse

    facilitation of field EPSP (fEPSP) amplitudes was enhanced indicating 5 containing

    GABAA receptors play a key role in cognitive processes by controlling a component of

    synaptic transmission in the CA1 region of the hippocampus.

    Requirement of 5 GABAA receptors for the development of tolerance to the sedative

    action of diazepam in knock-in mice, in which the 1, 2, 3, or 5 GABAA receptors had

    been rendered insensitive to diazepam by histidine-arginine point (van Rijnsoever et al.,

    2004). A reduction in 5 subunit-containing gamma-aminobutyric acid GABAA receptors

    has been reported to enhance some forms of learning in mutant mouse models (Yee et al.,

    2004). Moreover, the largely extrasynaptic 5 GABAA receptors in hippocampal

    pyramidal cells are implicated as control elements of the temporal association of threat

    cues in trace fear conditioning (Crestani et al., 2002).

    Wild type, 2 (H101R), and 3 (H126R) mice showed a robust diminution of the motor-

    depressant drug action. In contrast, 5 (H105R) mice failed to display any sedative

    tolerance. 1 (H101R) mice showed no alteration of motor activity with chronic

    diazepam treatment. Thus, the chronic activation of 5 GABAA receptors is crucial for the

    normal development of sedative tolerance to diazepam, which manifests itself in

    conjunction with 1 GABAA receptors. To identify the molecular and neuronal target

    mediating the anxiolytic action of benzodiazepines, (Low et al., 2000) generated and

    analyzed two mouse lines in which the 2 or 3 GABAA receptors, respectively, were

    rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of

    diazepam was absent in mice with the 2 (H101R) point mutation but present in mice

    with the 3 (H126R) point mutation. These findings indicate that the anxiolytic effect of

    benzodiazepine drugs is mediated by 2 GABAA receptors, which are largely expressed

    in the limbic system, but not by 3 GABAA receptors, which predominate in the reticular

    activating system. In another study it was shown that by introducing a histidine-to-

  • Chapter 1 Introduction

    17

    arginine point mutation at position 101 of the murine alpha1-subunit gene, that 1-type

    GABAA receptors, are rendered insensitive to allosteric modulation by benzodiazepine-

    site ligands, whilst regulation by the physiological neurotransmitter gamma-aminobutyric

    acid is preserved (Rudolph et al., 1999). Alpha1(H101R) mice failed to show the

    sedative, amnesic and partly the anticonvulsant action of diazepam. In contrast, the

    anxiolytic-like, myorelaxant, motor-impairing and ethanol-potentiating effects were fully

    retained, and are attributed to the nonmutated GABAA receptors found in the limbic

    system (2, 5), in monoaminergic neurons (3) and in motoneurons (2, 5).

    1.2: Histamine-receptors and the histaminergic system

    1.2.1: Histamine in the nervous system

    Histamine is one of the aminergic neurotransmitters, playing an important role in the

    regulation of several physiological processes. Histamine is synthesized and transported to

    brains of almost all animal species. The content of histamine varies between species,

    being higher in lower vertebrates and to be a lower level in mammals (Reite, 1972;

    Almeida and Beaven, 1981). Histamine containing nerve cells in the brain are found

    exclusively in the tubomamillary nucleus of the hypothalamus (TM nucleus) and they

    project throughout the brain and to all fields of hippocampus (Schwartz et al., 1991). In

    all mammals, the cerebral cortex, amygdala, substantia niagra and striatum receive

    moderate or dense histaminergic innervations. The density of projections in the

    hippocampus and thalamus varies, and the retina and spinal cord also receive

    histaminergic fibers from the TM nucleus. Also, afferent projections to TM neurons are

    wide spread and come from prominent sources like infralimbic cortex, lateral septum and

    preoptic nucleus (Ericson et al., 1991). The brain stem innervations in to TM nucleus

    include, the adrenergic cell group C1-C3, from noradrenergic groups A1-A3, and from

    serotonergic group B5-B9 also, only few fibers from locus coeruleus and the

    dopaminergic groups of substantia nigra and ventral tegmentum innervates TM nucleus.

  • Chapter 1 Introduction

    18

    Fig. 1.6: Distribution of histaminergic neurons in the brain. The histamine-producing neurons, located in the tuberomamillary nucleus of the human brain, innervate all of the major parts of the cerebrum, cerebellum, posterior pituitary and the spinal cord. Adapted from (Haas and Panula, 2003).

    Histamine is synthesized from histidine, which is transported in to neurons by L-amino

    acid transporter. Histidine decarboxylase converts histidine in to histamine. Histamine is

    then taken up in to vesicles by the vesicular monoamine transporter VMAT-2. After

    release into the synaptic cleft, histamine is methylated by histamine methyltransferase

    which is located postsynaptically and in glia to tele-methylhistamine (t-MHA), a

    metabolite that does not show any histamine like activity (Haas and Panula, 2003).

    1.2.2: Metabotropic histamine receptors

    Histamine is a ubiquitous chemical messenger that exerts numerous functions mediated

    through at least four pharmacologically distinct receptors (H1-H4), which are all members

    of the G-protein-coupled receptor family (Hill et al., 1997).

  • Chapter 1 Introduction

    19

    Fig. 1.7: Metabolism of Histamine in the neurons The L-amino-acid transporter brings histidine into neurons where histamine is synthesized by the specific enzyme histidien decarboxylase. Histamine is then taken up into vesicles by the vesicular monoamine-transporter VMAT-2. After release, histamine is methylated by histamine-methyltransferase which is located postsynaptically and in glia to tele-methylhistamine, a metabolite that does not show any histamine-like activity. Adapted from (Haas and Panula 2003).

    The H1 receptor is expressed in the brain, endothelial cells, and smooth muscle cells. The

    most characteristic roles for H1 receptor activation are smooth muscle contraction and

    increases in vascular permeability (Ash and Schild, 1966). The H1 receptor is a 486-491

    amino acid protein encoded by an intronless gene (Yamashita et al., 1991). H1 receptors

    mediate excitatory actions on whole brain activity. At the cellular level excitation is

    achieved by the activation of the Gq/11 heterotrimeric G-protein and its downstream

    effector phospholipase C (PLC). Stimulation of the Gq/11 - PLC pathway by the H1-

    receptor results in the synthesis of inositol-1, 4,5-trisphosphate and 1,2-diacylglycerol,

    which in turn stimulate an increase in intracellular Ca2+ and the activation of protein

    kinase C (PKC). H1 receptor activation can lead to activation of several other signaling

    pathways like stimulation of nitric oxide synthase activity (via a Ca2+/calmodulin-

    dependent pathway) and subsequent activation of soluble guanylyl cyclase in a variety of

    different cell types (Leurs et al., 1991; Casale et al., 1985; Duncan et al., 1980).

  • Chapter 1 Introduction

    20

    The H2 receptor was first cloned from dog and later found in several species. H2 receptor

    is an intronless gene and protein consists of 358-359 amino acids. The H2 receptor has

    been demonstrated to function as a key modulator for gastric acid secretion, and H2

    receptor antagonists are widely used for the treatment of gastrointestinal ulcers (Soll and

    Walsh, 1979). The direct action on neuronal membranes is usually excitatory or

    potentiates excitation. The H2 receptors signals through Gs-G-proteins, adenylyl cyclase

    and PKA, which phosphorylates proteins and activates the transcription factor cyclic-

    AMP response element binding protein.

    The H3 receptor was first characterized as an auto-receptor - regulating histamine

    synthesis and release from rat cerebral cortex, striatum, and hippocampus (Arrang et al.,

    1983, 1985). H3-receptor-mediated inhibition of histamine release has also been observed

    in human cerebral cortex (Arrang et al., 1988). H3 receptor is located presynaptically on

    histaminergic neurons. By alternative splicing several isoform of H3 receptors, consisting

    of 326-445 amino acids, are derived from a single gene. H3 also provides negative

    feedback to the release of other transmitter such as glutamate, acetylcholine and

    noradrenaline. H3 receptors are coupled to Gi\o and high voltage activated Ca2+ channels.

    The H3 receptors are coupled negatively by cAMP and activates the mitogen activated

    protein kinase pathways (Drutel et al., 2001).

    The H4 receptor is detected predominantly in the periphery, for example in bone marrow

    and leucocytes. The amino acid sequence of the H4 receptor has a 35 % amino acid

    homology with the H3 receptor and a much lower homology to H1 and H2 receptors. Very

    little is known about the actual biological function of H4 receptor. The H4 receptor can

    mediate chemotaxis and calcium influx in mast calls and eosinophils (O'Reilly et al.,

    2002; Hofstra et al., 2003).

  • Chapter 1 Introduction

    21

    Fig. 1.8: Signal transduction pathways mediated by various metabotropic histamine receptors. Histamine receptors and their coupling to G-proteins are shown on the left; the membrane targets of receptor activation are on the right. The box contains the signaling pathways and intracellular targets. AA, arachdonic acid; AC, adenylyl cyclase; CREB, cyclic-AMP-response element (CRE)-binding protein; DAG, diacylglycerol; GC, guanylyl cyclase, HVCC, high voltage activated Ca2+ -current; Ih, hyperploraization-activated cationic current; IAHP, the small conductance Ca2+-dependent K+ current; IK,leak, leak K+ current; Ins(1,4,5)P3, inositol-1,4,5-triphosphate; MAPK, mitogen activated protein kinase; NCX, Na+-Ca2+ exchanger; NMDA, N-methyl-d-aspartate; NO, nitric oxide; PKA, protein kinase C; PKC protein kinase C; PLA, Phospholipase A; PLC, Phospholipase C. Adapted from (Haas and Panula, 2003).

  • Chapter 1 Introduction

    22

    1.2.3: Interaction of histamine antagonists with GABAA receptors

    In one report the interaction of histamine H2 receptor antagonists with GABA and

    benzodiazepine binding sites in the CNS was analyzed (Lakoski et al., 1983). The

    histamine H2-receptor antagonist cimetidine potently inhibited [H3] muscimol and

    enhanced [H3] flunitrazepam binding in membranes prepared from several brain regions

    in the rat, including the dorsal raphe nucleus. As further examined in cortical membranes,

    this effect on both GABA and benzodiazepine binding sites was specific for imidazole-

    derived H2 receptor antagonists (potency: cimetidine greater than metiamide greater than

    tiotidine) and not observed with either several H1 receptor antagonists or histamine. Their

    data indicate a striking similarity between the actions of cimetidine (and other imidazole-

    derived H2 receptor antagonists) and GABA on binding parameters at the GABA receptor

    complex.

    In one report the in vitro antagonism of benzodizepines binding to cerebral receptors by

    H1 and H2 histamine antagonists was checked (Speeg et al., 1981). They investigated

    about the depressant action of histamine antagonists in CNS. It was demonstrated that

    cimetidine and pyrilamine are competitive antagonists of 3H-benzodiazepine binding to

    human cerebral receptor in vitro. Therefore, the interaction of antihistamine with CNS

    receptors other than histamine receptor may explain, at least in part, the side effect of

    sedation.

    1.2.4: Ionotropic histamine receptors and direct modulatory actions of histamine to

    ion channels

    In another study the effect of histamine H2-receptor antagonists on the GABA-responses

    of the intestine was investigated. GABA and the GABAA agonist muscimol were applied

    to isolated ileal guinea pig preparations in the absence and presence of two H2 receptor

    antagonists, famotidine and cimetidine. Both GABA and muscimol produced a

    concentration-dependent contractile effect on the guinea pig ileum. Famotidine and

    cimetidine modified this contractile effect, either by enhancing or by inhibiting it. The

  • Chapter 1 Introduction

    23

    differing results depended not only on the antagonist concentration, but also on the

    concentration of GABA or muscimol. In conclusion, the interaction of H2 receptor

    antagonists with GABA receptors is not limited to the central nervous system, but it also

    extends to the peripheral nervous system. The receptor interaction mainly involves

    GABAA receptors and depends on both the specific H2 antagonist and the concentration

    used (Koutsoviti-Papadopoulou et al., 2003).

    To test the hypothesis that cimetidine-like drugs produce CNS effect like seizure and

    analgesia effects via inhibition of GABAA receptors, the actions of these drugs were

    studied. The H2 antagonists famotidine and tiotidine produced competitive and reversible

    inhibition of GABA-evoked currents in HEK 293 cells transfected. In contrast, the H2

    antagonist ranitidine and the cimetidine congener improgan had very weak (if any)

    effects. Authors concluded that cimetidine-like drugs do not appear to produce seizures

    or analgesia by directly inhibiting GABAA receptors (Cannon et al., 2004).

    In contrast to the multiple genes for metabotropic histamine receptors, no genes for

    ionotropic histamine receptors have been identified in mammals up to now. There are few

    hints from electrophysiological experiments that in mammals such direct activated

    channels may also exist and that histamine mediate fast synaptic inhibition of rat

    supraoptic oxytocin neurons via chloride conductance activation. Up to now, the ion

    channels mediating this action were not identified (Hatton and Yang, 2001). At N-

    methyl-D-aspartate (NMDA) receptors, histamine enhances the glutamate-evoked current

    by direct binding to the channel protein itself. Histamine causes a direct facilitation of the

    NMDA-receptor through its polyamine modulatory sites. When applied to cultured

    hippocampal neurons, histamine selectively increased by up to tenfold the amplitude of

    the component of synaptic transmission that was mediated by NMDA-receptor (Bekkers,

    1993). By selectively enhancing the NMDA component of neurotransmission, histamine

    should enhance process in which NMDA currents participate, such as triggering of Long-

    term potentiation. Conversely, pathological conditions that deplete histamine in the brain

    might lead to a reduced ability to trigger Long-term potentiation and so to memory loss.

  • Chapter 1 Introduction

    24

    In insects, histamine-acivated chloride channels were known for a long time. Native

    ionotropic histamine receptors of invertebrates have been characterized in vivo,

    particularly in the large monopolar neurons of the visual system of Drosophila (Hardie,

    1989), the heart ganglion (Hashemzadeh-Gargari and Freschi, 1992), and the olfactory

    receptor neurons of lobster (McClintock and Ache, 1989), where they mediate the pre-

    synaptic inhibition of ORNs (Wachowiak et al., 2002). The ionotropic histamine

    receptors mediate rapid neurotransmission in the visual system of invertebrates (Burg et

    al., 1993; Hardie, 1989). Recently, genes for histamine-gated ion channels were

    identified (Zheng et al., 2002; Gisselmann et al., 2002). Two histamine receptor subunits

    have been so far cloned from Drosophila: HisCl-1 (alias hisCl2, ort, hclA and Dm-

    HACL1) and HisCl2 (alias hisCl1, ort, hclB and Dm-HACL2) (Gengs et al., 2002;

    Zheng et al., 2002; Gisselmann et al., 2002). Both form homomultimeric chloride

    channels when expressed in Xenopus oocytes, where HisCl-2 is about an order of

    magnitude more sensitive than HisCl- (Zheng et al., 2002; Gisselmann et al., 2002).

    1.2.5: Histamine functions and knockout mice

    The histaminergic neurons are involved in many functions such as memory, sleep, and

    alertness and feeding. Histaminergic neurons send widespread projections to most

    cerebral regions, including those known to be important in sleep-wake control, such as

    the cortex, thalamus, and posterior and preoptic / anterior hypothalamus, and to the

    forebrain and brainstem aminergic and cholinergic structures (Inagaki et al., 1988; Panula

    et al., 1989). In these target areas, histamine modulates neuronal activity-excitability via

    H1, H2, and H3 receptors. Moreover, histaminergic neurons firing rate varies across the

    sleep-wake cycle, being highest during waking and lowest during rapid-eye movement

    sleep.

    Administration of various substances impairing histaminergic transmission increases slow

    wave sleep, whereas enhancement of transmission promotes wakefulness (Monti et al.,

    1991). Muscimol-induced inactivation of the posterior hypothalamus containing

    histaminergic cells results in hypersomnia in both normal and experimentally induced

  • Chapter 1 Introduction

    25

    insomniac cats (Lin, 2000). Finally, inhibition of histamine synthesis in the same area

    increases slow wave sleep, whereas inhibition of histamine degradation elicits long-

    lasting arousal (Lin et al., 1986, 1988). In histidine decarboxylase knockout mice,

    disruption of histamine synthesis causes permanent changes in the cortical EEG and

    sleep-wake cycle and that, at moments when high vigilance is required (lights off,

    environmental change etc.), mice lacking brain histamine are unable to remain awake

    (Parmentier et al., 2002). Neuronal histamine has been shown to suppress food intake

    through activation of histamine H1 receptors in the ventromedial hypothalamus or

    inhibition of the H3 receptor in the paraventricular nucleus (Sakata et al., 1988; Ookuma

    et al., 1989) each of which is involved in satiety regulation.

    Leptin, an ob gene product (Zhang et al., 1994) has been demonstrated to promote

    histamine turnover by affecting the post - transcriptional process of histidine

    decarboxylase formation or histamine release per se (Yoshimatsu et al., 1999). In

    addition, concentration or turnover rate of hypothalamic histamine was lowered in leptin-

    deficient ob/ob and leptin receptor-mutated db/db mice, but it was increased in diet-

    induced obese animals (Yoshimatsu et al., 1999). In H1 receptor knockout mice it has

    been shown that H1 - receptor is a key receptor for downstream signaling of leptin in the

    brain that contributes to regulation of feeding, fat deposition, and UCP mRNA expression

    (Masaki et al., 2001). Histamine also alters thermoregulation; hypothalamic

    histaminergic neurons are activated not only peripherally by high ambient temperature,

    but also centrally by Interleukin L-1beta as endogenous pyrogen (Kang et al., 1994). H3 receptor knockout mice display reduced locomotion and body temperature (Toyota et al.,

    2002). Histamine neurons stimulate the sympathetic nervous system to increase lipolysis

    in the adipose tissue (Bugajski and Janusz, 1981) an effect that depends more on H1

    receptor than H2. Also, Stimulation of supraoptic nucleus by histamine causes synthesis

    and release of vasopressin which in turn induces antidiuresis (Haas et al., 1975;

    Armstrong and Sladek, 1985; Tuomisto et al., 1980).

  • Chapter 1 Introduction

    26

    1.2.6: Diseases where histamine is involved

    Histamine is assumed to be involved in neurodegenerative disorders like in Alzheimer.

    Numerous neurofibrillary tangles were found in the Alzheimer hypothalami, concentrated

    in the tuberomammillary area. Most of them were of globular type and extracellular, and

    only a minority were histamine immunoreactive. They may represent remnants of

    degenerated TM (Nakamura, 1993). Decrease in brain histamine as well as histidine may

    contribute to the cognitive decline in Alzheimer's disease directly or through the

    cholinergic system (Schneider et al., 1997). The TM neurons seem morphologically

    normal in patients with Parkinson disease though, the central histaminergic system

    appears to be activated in Parkinson disease, and since the histaminergic innervation is

    increased in the substantia nigra. Also, modulation of the histamine H3 receptor occurs in

    Parkinson disease at the level of the mRNA expression in the striatum and receptor

    density in the substantia nigra. Marked increase occurs in histamine H3 receptors in the

    striatum and substantia nigra by tonic dopaminergic inputs (Ryu et al., 1994).

    There is growing evidence to suggest the involvement of histaminergic pathways in the

    pathophysiology of schizophrenia. In agreement, decreased H1 receptor-mediated

    response to histamine is consistently observed among schizophrenic patients (Rauscher et

    al., 1980; Nakai et al., 1991). Levels of t-MHA, the major histamine metabolite in brain

    (Schwartz et al., 1971) are significantly enhanced in the cerebrospinal fluid of

    schizophrenic patients (Prell et al., 1995). Finally, a polymorphism within the H2 receptor

    gene was recently reported to be associated with schizophrenia (Orange et al., 1996).

    Many patients diagnosed as schizophrenic have either a chronic excess or deficiency of

    blood histamine. Nutritional treatment correcting these imbalances has led to great

    improvement or recovery for most such patients. Histamine is used to promote alpha

    wave activity in the brain, which enables an individual to handle anxiety and stress easier

    (McLeod et al., 1998). If the person is deficient in histidine, it leads to a lack of histamine

    and creates unbalances in calming alpha-rhythms in the brain allowing the excitatory beta

    waves (responsible for the brain activity that leads to anger and tension to promote)

    (McLeod et al., 1998).

  • Chapter 1 Introduction

    27

    1.2.7: Aims of the work

    In Insects, the existence of histamine-gated chloride channels is long known. The

    possible occurrence of such channels in vertebrates has been long postulated but no gene

    was identified until now. Such channels have a fair chance to belong to the gene-family

    of ligand- gated channels.

    There were some indications that a so far undiscovered correlation between histamine

    and GABA on the level of receptors exist. In insects, GABA and histamine gate the same

    channel. In mammals, GABAA receptors are co-localized in close proximity to

    histaminergic neurons, but specific interrelationship between GABA and histamine has

    not been investigated yet. Therefore the aims of my work were to identify possible

    candidates with bioinformatical means for histamine-gated or modulated channels in

    vertebrates and to check especially members of the class of ligand-gated ion channels for

    possible genes with similarity to insect histamine-gated channels.

    The cDNA of found candidates should be cloned and functionally expressed in Xenopus

    oocytes. The action of histamine should then be characterized by a two-electrode voltage

    clamp measurements.

  • Chapter 2 Materials

    28

    Chapter 2

    2: Materials

    2.1: Chemicals and enzymes

    Agarose LE, analytical grade, Biozym

    Albumine, bovine, Fraction V, Sigma,

    Alkaline Phosphatase, Shrimp, Roche

    Ampicillin Disodium Salt, Sigma

    ATP, Disodium Salt, Sigma

    Collagenase, Worthington Biochemical Corporation

    DMSO, Sigma

    dNTPs, Invitrogen

    DTT, Invitrogen

    Diethyl Pyrocarbonate, Sigma

    EDTA Disodium Salt, Sigma

    Ethidium Bromide solution, Sigma

    Fetal Bovine Serum, Invitrogen

    Formamide, Sigma

    Goat serum, Gibco, Sigma

    Herring Sperm DNA, Roche

    Levamisole, Sigma

    Proteinase K, Roche

    Restriction enzymes from: MBI Fermentas, Roche

    RNase A, pancreatic, Roche

    RNase H, Roche

    RNasin, MBI Fermentas

    RNase-free DNaseI, Roche, Biozym

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    SUPERSCRIPT III RNase H- Reverse Transcriptase, Invitrogen

    T4 DNA ligase, MBI Fermentas

    T4 DNA polymerase, MBI Fermentas

    T4 polynucleotide Kinase, MBI Fermentas

    Taq DNA Polymerase, Invitrogen

    All other standard chemicals were from Sigma, Fluka, Aldrich Baker, Gerbu, Merck,

    Pharmacia, Promega, Riedel de Haen, Roth and Serva and used typically in p.a. quality.

    2.2: Drugs used for pharmacological characterizations

    Stocks solutions of drugs used for pharmacological characterizations were prepared as

    indicated in the following list. If Xenopus Ringer was used as the solvent, care was taken

    to check and if necessary adjust the pH to 7.4 after solving of the drugs as especially

    histamine acidifies the agonist solutions.

    Neurotransmitters:

    Acetylcholine 100 mM Xenopus Ringer Sigma

    ATP 100 mM Xenopus Ringer Sigma

    Dopamine 100 mM Xenopus Ringer Sigma

    GABA 1 M Xenopus Ringer Sigma

    Glycine 1 M Xenopus Ringer Sigma

    Glutamate 1 M Xenopus Ringer Sigma

    Histamine 1 M Xenopus Ringer Sigma

    Octopamine 100 mM Xenopus Ringer Sigma

    Serotonin 10 mM Xenopus Ringer Sigma

    GABA-receptor related drugs:

    Bemegride 300 mM DMSO Acros

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    Diazepam 100 mM DMSO Roche

    Pentobarbital 10 mM DMSO Sigma

    Propofol 50 mM DMSO Tocris

    Flunitrazepam 1 mM Xenopus Ringer Ratiopharm

    Histamine-receptor related drugs:

    Doxylamine 10 mM DMSO RBI

    DM235 100 mM DMSO Sigma

    Cimetidine 10 mM Xenopus Ringer Sigma

    Famotidine 10 mM Xenopus Ringer Wallgreen's

    HTMT 30 mM DMSO Tocris

    Histidine 100 mM Xenopus Ringer Sigma

    Pyrilamine 100 mM DMSO RBI

    R-alpha-Methylhistamine 100 mM Xenopus Ringer Tocris

    tele-Methylhistamine 30 mM Xenopus Ringer RBI

    Thioperamide 30 mM Xenopus Ringer Tocris

    Others:

    Harmane 100 mM DMSO Tocris

    PTX 3 mM Xenopus Ringer Sigma

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    2.3: Primers

    All oligonucleotides (primers) were purchased from Invitrogen or MWG Biotech and solved in H2O to a concentration of 100 pmol / l. Primers S.No. Subunit Primer Sequence

    1 hGABA-th Not1-Stop GAC TGC GGC CGC TTA ATC GAT ATA CAT ATG GTA TAC CCA

    2 r GABA-th fw GCC ATC CAC ATT ACT GAC GAG CTA CAC 3 r GABA-th.5-new GCC GAA TTC GCC ATG GGC ATC CGA GGC ATC CGA

    GGC ATG CTG 4 Gaba-t-Eco.ATG GAC GAA TTC CAC CAT GCT GCG AGC CGC TGA GCT

    CCT 5 m GABAth-3 GCA TGG GCC CTA ATA GAC ATG GTA TAA CCA 6 r GABAth-rev TAT CAG GCC ATC CTG CAC ATG TGC TAC 7 Gaba-t-Cla1-sp-Xho1 CTC GAG CTA ATC GAT ATA CAT ATG GTA TAA CCA

    GTA 8 mGABA-th-fw GCA TGC GGC CGC CAT CCA CAT TAC TGA TGG GCT G 9 r GABA th-1000-fw GGC TCG AGC TCC TGG ATA TCA TTT TGG ATG 10 mGABA th-rev GCC ATG TGA ACA CCA AGG ATC CTA GAC 11 rGAB-th-3-new ATC CTC GAG CCT GCT GCT GTG GTG ATA CTC 12

    mGABA-th-5 rGABAth-3-new

    GCA TGC GGC CGC CAT GGG CAT CCG AGG TAT GCT G ATC CTC GAG CCT GCT GCT GTG GTG ATA CTC

    13 rGe-EV-r ATC ATA CTC TTG GGT CCT CTT AGA ATT CC 14 rGAe-up GCT GAG ATG TTG CCT AAA GTT CTC C 15 rG eEV ATA ACC ATA CCC AAC CAG ATG GC 16 Mus-rat-gaba-e-up ACC ATG GTG CCT AAA GTT CTC CTG ATG 17 Mus-rat-gaba-e-down CCA GCT GGA GCC TAC AGG TTA AGG 18 r GA e-down TCC TGG GGA ACT GAG GTG ATT GC 19 1 H-B1-rev GTG TAC ATA GTA AAG CCA ATA AAC 20 1 r GABA b1-down GAG TCT AA CCG AAC CAT GAG AC 21 1 H-B1-fw TGG ACA GTA CAA AAT CGA GAG AG 22 1 r-B1 fw TGG ACA GTA CAA AAT CGA GAG AGT TTG 23 1 r-B1-rev GTG TAC ATA GTA AAG CCA ATA AAC GA 24 2 RR-GABA B2-up GCC ACC ATG TGG GGC TTT GCG GGA GGA AGG 25 2 RR-GABA- B2-Do ATC AAG TGT TAA CAT AGT ACA GCC AG 26 3 r-GABA-B3-Del Stop GTT AAC ATA GTA CAG CCA GTA AAC TAA 27 2 r-GABA g2-atg-BamH1 CCG GAT CCA CCA TGA GTT CGC CAA ATA CAT GG 28 1 r-GA-a1-4 CAA GCC CGT GAT GAA GAA AAG TCG Stop codon:

    29 3 H-GABA b3 Not1-Stop EV

    GAC TGC GGC CGC TAG ATA TCG TTA ACA TAG TAC AGC CAG TA

    30 2 H-GABA g2-Not 1-Stop-EV GAC TGC GGC CGT TAG ATA TCC AGA TAA AGA TAG GAG ACC CA

    31 1 GABA a1-Not1-Stop-Ev GAC TGC GGC CGC TAG ATA TCT TGA TGG GGT GTG GGG

    32

    2 H-GABAA g2- Not 1-Stop EV

    GAC TGC GGC CGC TAG ATA TCC AGA TAA AGA TAG GAG ACC CA

    33 2 rGABAg2-Stop-Xho1 GCC TCG AGT CAC AGA TAA AGA TAG GAG AC 34 rGABA-e-Not1-StopEV GAC TGC GGC CGC TAG ATA TCC AGG TTA AGG CAA

    ATC ACC CAG TA

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    35 HGABA-e-rev-O CAA GTT AAG GCA AAC AAG CCA GTA GAG CAC 36 HGABAe-fw-O TTG TCG AAA GTT CTT CCA GTC TTC CTA GGC 37 RGABA-e-fw-O TTG CCT AA GTT CTC CTG ATG CTC CTC 38 RGABA-e-rev-O CAG GTT AAG GCA AAT CAC CCA GTA GAC 39 Rat-ep-start-H3-r CTG GAA GCT TCT TTC CAC TAG GCT GAG GCT GAG

    GCC CAA AG 40 GFP 1\2 EcoRV-GFP-5` ATC GTG AGC AAG GGC GAG GAG CTG TTC ACC 41 GFP GFP down w\o stop ATC CTT GTA CAG CTC GTC CAT GCC

    2.4: Standards for DNA

    Following pre-made DNA size standards were used:

    Gene Ruler 100 bp DNA Ladder, MBI Fermentas

    Gene Ruler 1 Kbp DNA Ladder, MBI Fermentas 250 bp DNA Leiter, Diagonal

    2.5: Consumption materials

    Borosilicate glass capillaries (GC150TF-10) for electrophysiological studies were from

    Clark Electronical Instruments. Pipette tips were from Sarstaed. Other plastic ware like

    15 ml and 50 ml polypropylene tubes, 50 ml plastic tubes, 0.5-1.5 ml reaction tubes as

    well as PCR-tubes, syringes and Petri dishes were bought from Sarstaed, Eppendorf or

    Biozym. Cell culture dishes were from Nunc.

    Distilled water was prepared in a Quarz-double distilling unit and autoclaved at 121C

    and 20 PSI for 20 minutes. Plastic ware that had to be sterile was either autoclaved under

    the same conditions for 15 minutes or was used from unopened bags.

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    2.6: Kits

    RNA isolation:

    TRIzol Reagent, Invitrogen

    DNA cleanup and isolation:

    QIAquick PCR Purification Kit, Qiagen,

    QIAquick Nucleotide Removal Kit, Qiagen,

    QIAquick Gel Extraction Kit, QiagenPlasmid DNA isolation

    Qiagen plasmid maxi Kits, Qiagen

    Others:

    Amplicap T7 or T3 Kit, Biozyme

    First strand synthesis: SuperScript III, Invitrogen

    2.7: RNase free materials and chemicals

    When working with RNA only RNase free solutions and vessels are used, to avoid

    degradation of RNA.

    For the production of RNase free water (Sambrook et al., 1989), ddH2O water was

    combined with DEPC to a final concentration of 0.1 %, incubated for 30 minutes at room

    temperature and then autoclaved for 15 minutes at 20 PSI and 121 C to expel DEPC.

    DEPC destroys RNases, but can also kill RNA. However, when it comes in contact with

    moisture, it hydrolyzes to form ethanol and carbon dioxide and is therefore rendered

    harmless through appropriate treatment.

    Glassware was baked over night at 180 C in an oven and solutions were made with

    DEPC-water in those RNase free vessels.

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    2.8: Frequently used buffers

    Agar-plates: 15 g/l in LB-/NZ-Medium,

    autoclaved

    Alkaline Phosphatase-buffer, 10 x: 500 mM Tris-HCl; pH 8.5

    50 mM MgCl2

    Ampicillin stock: 10 mg/ml in H2O

    Barths solution: 88 mM NaCl

    1 mM KCl

    0.82 mM MgSO4

    0.33 mM Ca(NO3)2

    0.41 mM CaCl2

    2.4 mM NaHCO3

    5 mM Tris-HCl, pH 7.4

    Ethidiumbromide stock: 10 mM in H2O

    HBS, 10 x: 1.4 M NaCl

    0.25 M HEPES

    14 mM Na2HPO4

    LB (Luria-Bertani) medium; pH 7.4: 10 g/l Tryptone

    5 g/l yeast extract

    10 g/l NaCl

    autoclaved

    Ligation buffer, 5 x: 250 mM Tris-HCl; pH 7.6

    50 mM MgCl2

    5 mM ATP

    5 mM DTT

    Lysis buffer: 10 mM Tris-HCl; pH 7.9

    1 mM EDTA

    15 % Sucrose

    2 mg/ml Lysozyme

    0.2 mg/ml RNase

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    0.1 mg/ml BSA

    M10-Medium: 500 ml MEM

    50 ml FBS

    5 ml L-Glutamine, 200 mM

    5 ml Pen/Strep-Soln.

    ND96 99.6 mM NaCl

    2 mM KCl

    1 mM MgCl2

    5 mM HEPES, pH 7.5

    Pen/Strep solution: 10,000 U Penicilline

    10 mg Streptomycine

    in 1 ml 150 mM NaCl soln.

    Probe buffer, 5 x: 20 % Ficoll 400, (w/v)

    100 mM EDTA

    0,025 % Bromephenolblue

    0,025 % Xylenecyanole, (w/v)

    PNK buffer: 250 mM Tris-HCl; pH 7.6

    50 mM MgCl2

    25 mM DTT

    0,5 mM Spermidine

    0,5 mM EDTA

    Buffer P1: 50 mM Tris-HCl; pH 7.9

    10 mM EDTA

    100 g/ml RNase A

    Buffer P2: 200 mM NaOH

    1 % SDS

    Buffer P3: 3 M Potassium acetate; pH 7.4

    Buffer QBT: 750 mM NaCl

    50 mM MOPS; pH 7.0

    15 % Ethanol (v/v)

    0.15 % Triton X-100, (v/v)

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    Buffer QC: 1 M NaCl

    0.05 M MOPS; pH 7.0

    15 % Ethanol

    Buffer QF: 1.25 M NaCl

    0.05 M Tris-HCl; pH 8.5

    15 % Ethanol (v/v)

    Reverse Transcriptase buffer, 5x 250 mM Tris acetate, pH 8.4

    375 mM Potassium acetate

    40 mM Magnesium acetate

    1.-Strand buffer, 5 x: 250 mM Tris-HCl, pH 8.3

    375 mM KCl

    15 mM MgCl2

    Taq-DNA-Polymerase buffer, 10 x: 200 mM Tris-HCl; pH 8.4

    500 mM KCl

    TBE: 90 mM Tris-HCl; pH 8.3

    90 mM Boric acid

    2 mM EDTA

    TE, 1 x: 10 mM Tris-HCl; pH 7.9

    1 mM EDTA

    Transcription buffer: 400 mM Tris-HCl; pH 8.0

    60 mM MgCl2

    100 mM Dithiothreitol

    20 mM Spermidine

    Xenopus Ringer's soln. 115 mM NaCl

    2.5 mM KCl

    1.8 mM CaCl2

    10 mM Hepes, pH 7.2

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    2.9: Bacterial strains

    XL1-Blue (Stratagene) recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1

    lac [FproAB lacIqZM15 Tn10 (Tetr)]c

    2.10: Plasmid vectors

    General cloning and expression vectors: pSGEM: Oocyte expression vector derived from pGEMHE vector (3022 bp); (Liman et

    al., 1992) Kindly provided by Dr. Michael Hollmann, Bochum.

    pBluescript II KS (+) (Stratagene)

    pRC/CMV (Invitrogen)

    pCDNA3 (Invitrogen)

    GABA-receptor expression vectors:

    Following vectors originate from the German Genome Resource Center Berlin (RZPD):

    GABAA-receptors:

    Human delta subunit in pCMV-SPORT6 clone IRATp670E0653D6

    Human alpha 1 subunit in pBluescriptR clone IRAKp961A1533Q

    Human alpha 3 subunit in pBluescriptR clone IRAKp961F0547Q

    Human alpha 5 subunit in pT3T7 clone IRATp970H0853D6

    Human alpha 6 subunit in pSPORT6-sfi clone DKFZp686D23263Q

    Human beta 3 subunit in pCMV-SPORT6 clone IRAKp961K0613Q

    Human gamma 1 subunit in pBluesciptR clone IRAKp961J0448Q

    Human gamma 2L subunit in pCMV-SPORT6 clone IRAKp961L0931Q

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    GABAC-receptors:

    Human rho1 subunit in pT3T7-PacI clone IMAGp998P2111525Q

    The plasmid pCDNA3.1-GABA-myc-theta containing the human GABAA-theta subunit

    cDNA was a kindly gift of P. Wingrove, MSD. The plasmid pCDNA-GABA-2

    containing the rat GABAA-2 subunit cDNA was kindly provided by R. Ruprecht,

    Mnchen.

    The plasmids:

    pCDNA3-rGA1 containing the rat GABA(A) alpha1-subunit cDNA in pCDNA3

    pSGEM-rGA1 containing the rat GABA(A) alpha1-subunit cDNA in pSGEM

    pCDNA3-rGA1 containing the rat GABA(A) beta1-subunit cDNA in pCDNA3

    pSGEM-rGA1 containing the rat GABA(A) beta1-subunit cDNA in pSGEM

    pCDNA3-rGA containing the rat GABA(A) epsilon-subunit cDNA in pCDNA3

    pSGEM-rGA containing the rat GABA(A) epsilon-subunit cDNA in pSGEM

    were from the Lehrstuhl fr Zellphysiologie plasmid collection.

    2.11: Software

    General sequence analysis was done with the DNASTAR program package. Alignments

    of DNA sequences were done using the program Megalign. DNA sequences can be

    translated into amino acid sequences with Mapdraw, which also can find restriction sites

    for restriction enzymes. Protein as well as DNA Sequence comparisons to known and

    published sequences were performed in the Internet (NCBI USA) with BLAST (Basic

    Local Alignment Search Tool) Search, according to (Altschul et al., 1990).

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