DISSERTATION Titel der Dissertation VALERENIC ACID, ACTEIN AND DERIVATIVES: SUBUNIT- DEPENDENT MODULATION OF GABA A RECEPTORS Verfasserin Mag. pharm. Sophia Khom angestrebter akademischer Grad Doktor der Naturwissenschaften (Dr. rer. nat) Wien, 2008 Studienkennzahl lt. A 449 091 Studienblatt: Dissertationsgebiet lt. Pharmazie Studienblatt: Betreuerin / Betreuer: Univ. Prof. Dr. Steffen Hering Table of Contents 1. Introduction / State of Research 1 1.1. Basis of neuronal excitability 1 1.2 γ-aminobutyric acid (GABA) 1 1.2.1 GABAergic neurons 2 1.2.2 GABAergic synapses 2 1.3 GABA type A (GABA A) receptors 3 1.4 GABA A receptor dynamics 6 1.4.1 Assembly of GABA A receptors 6 1.4.2 Targeting and regulation of membrane expression 7 1.4.3 Clustering of GABA A receptors at synapses 8 1.4.4 GABA A receptor internalization and recycling 10 1.5 Structural determinants of the agonist binding site 10 1.6 Gating of GABA A receptors 11 1.7 Structure of the ion permeation pathway 13 1.8 Structure of the selectivity filter 13 1.9 The gates 14 1.10 Distribution pattern of GABA A receptor subunits 15 2. Modulation of GABA A receptors 16 2.1 Benzodiazepines 16 2.1.1 The benzodiazepine binding site 17 2.1.2 Benzodiazepine mechanism of action 19 2.1.3 Effects of benzodiazepines are mediated by different GABA A receptor subtypes 21 2.1.3.1 GABA A receptors mediating sedative-hypnotic effects 22 2.1.3.2 GABA A receptors mediating amnesia 22 2.1.3.3 GABA A receptors mediating anticonvulsant effects of benzodiazepines 22 2.1.3.4 GABA A receptors mediating anxiolytic effects of benzodiazepines 23 2.1.3.5 GABA A receptors mediating myorelaxation 23 2.1.3.6 GABA A receptors mediating analgesic effects 23 2.1.4 Modulation of GABA A receptors containing γ1 subunits 24 2.2 Barbiturates 24 2.2.1 Direct activation of GABA A receptors by barbiturates 25 2.3 General and volatile anaesthetics 26 2 2.3.1 Etomidate 27 2.3.2 Propofol 29 2.3.3 Volatile anaesthetics 30 2.4 Neurosteroids 31 2.4.1 Influence of the subunit composition on neurosteroid action on GABA A receptors 32 2.4.2 The neurosteroid binding site 33 2.5 Ethanol 35 2.6 Cations modulating GABA A receptor function 36 2.7 Other modulators of GABA A receptors 38 2.7.1 Loreclezole 38 2.7.2 Mefenamic acid 38 2.7.3 Clozapine 38 2.7.4 Furosemide 39 3. Tonic and phasic inhibition 40 4. Pathophysiological conditions associated with impaired GABAergic neurotransmission 41 4.1 Insomnia 41 4.2 Anxiety 42 4.3 Epilepsy 42 4.3.1 Mutations in GABA A receptor subunits associated with epilepsy 43 4.3.2 Temporal Lobe Epilepsy 44 4.3.3 Absence Epilepsy 44 4.4 Schizophrenia 45 5. GABA B receptors 46 6. GABA C receptors, a GABA A receptor subtype 48 7. Modulation of GABA A receptors by plant extracts and natural products 49 7.1 Valerian ( Valeriana officinalis ) 49 7.2 Black Cohosh ( Actaea racemosa ) 50 8. Aims 51 9. Materials and Methods 52 9.1 Chemicals 52 9.2 Animals 52 9.3 Expression and functional characterization of GABA A receptors 53 9.4 Perfusion system 54 3 9.5 Analyzing concentration-response curves 55 9.6 Open Field test 55 9.7 Elevated Plus Maze test 55 9.8 Light-Dark Choice test 56 9.9 Stress-induced hyperthermia test 56 9.10 Home Cage Activity 56 9.11 Statistical analysis of behavioral experiments 56 10. Results 57 10.1 Valerenic acid potentiates and inhibits GABA A receptors: mechanism and subunit specifity 57 10.1.1 Modulation of I GABA by Valerian extracts 57 10.1.2 Potentiation of I GABA by valerenic acid through α1β2, α1β2γ1 and α1β2γ2S channels 58 10.1.3 Potentiation of I GABA by valerenic acid through α2β2, α3β2, α4β2γ2S , α5β2, α2β2γ1 and α2β2γ2S channels 59 10.1.4 Modulation of I GABA by valerenic acid at different GABA concentrations 62 10.1.5 Valerenic acid does not interact with the BZD site 62 10.1.6 β-subunit dependence of GABA A receptor potentiation by valerenic acid 63 10.1.7 Direct activation of GABA A receptors by valerenic acid 66 10.1.8 Evidence for open channel block by valerenic acid and acetoxy- valerenic acid 68 10.2 Actein- a novel highly efficient modulator of GABA A receptors with strong in vivo effects 71 10.2.1 Modulation of I GABA by Actaea racemosa extracts 71 10.2.2 Potentiation of I GABA by actein through GABA A channels composed of α1, β2 and γ2S subunits 72 10.2.3 α-subunit dependence of I GABA modulation by actein 72 10.2.4 Influence of different β-subunits on I GABA potentiation by actein 75 10.2.5 Modulation of I GABA by actein at different GABA concentrations 76 10.2.6 Performance in the Open Field test (OF) after actein administration 77 10.2.7 Performance in the Elevated Plus Maze (EPM) and Light-Dark Choice Test (LDT) after actein administration 78 10.2.8 Effect of actein on stress-induced-hyperthermia (SIH) 80 10.2.9 Home Cage Activity measured after actein administration 80 11. Discussion 82 11.1.1 Subunit specificity of I GABA stimulation by valerenic acid 82 4 11.1.2 Slow activation and deactivation of I VA 83 11.1.3 Evidence for open channel block 83 11.2 Modulation of GABA A receptors by Actaea racemosa extracts 85 11.2.1 Actein modulation of GABA A receptors composed of different α or β subunit variants 85 11.2.2 Actein modulates I GABA independent on the GABA concentration 86 11.2.3 In vivo effects of actein 86 12. Summary 89 13. Zusammenfassung 90 14. References 91 15. Acknowledgments 109 16.Curriculum vitae 110 5 1. INTRODUCTION / STATE OF RESEARCH 1.1. BASIS OF NEURONAL EXCITABILITY At the end of the 18 th century Galvani and Volta suggested that propagation of nerve impulses and muscle contraction are based on electrical signals. In 1902, Bernstein hypothesized that in nerve and muscle cells the unequal distribution of potassium (K +)- ions over the cell membrane and the selective K +-permeability produces the resting potential and that a transient breakdown of selective permeability induces excitation, allowing other ions to pass the membrane. These changes in ion permeability along the axon of a neuron were suggested to build the backbone of the propagation of nerve impulses. Isolation of the squid giant axon and subsequent electrophysiological experiments by Hodgkin and Huxley in 1952 revealed that ion movements are the basis for the propagation of nerve impulses and local changes of the membrane potential (Zubay, 2000). In the central nervous system (CNS) communication between neurons is based on both electrical and chemical means. A prerequisite for propagation of electrical signals are gap junctions and a cytoplasmatic continuum between presynapse and postsynapse. A communication based on chemical means (e.g. neurotransmitters or hormones) involves a ligand that travels the distance between the cell by diffusion and binds to specific proteins (Luddens and Korpi, 1995; Rang et al., 2003). 1.2 γγγ-AMINOBUTYRIC ACID (GABA) GABA (see structural formula in Fig. 1) is the major inhibitory neurotransmitter in the CNS of the mammals (Sieghart, 1995; Barnard et al., 1998; Sieghart, 2006). GABA is synthesized from glutamate catalysed by two isoforms of the glutamic acid decarboxylase (GAD) GAD 65 and GAD 67 (Erlander et al., 1991). GABA is stored in synaptic vesicles by vesicular neurotransmitter transporters and released from nerve terminals by calcium-dependent exocytosis (Owens and Kriegstein, 2002). There is also evidence for non-vesicular secretion of GABA by e.g. reverse transporter action, a mechanism that might be of particular relevance during brain development (Attwell et al., 1993; Taylor and Gordon-Weeks, 1991). GABA interacts with ionotropic GABA type A (GABA A) and metabotropic GABA type B (GABA B) receptors, which can be localized either pre- or postsynaptic (Bormann, 2000; Owens and Kriegstein, 2002). Reuptake of the neurotransmitter into nerve terminals and/or surrounding glial cells by the plasma-membrane bound GABA transporter (GAT) terminates the action of the neurotransmitter (Cherubini and Conti, 2001). GABA is finally metabolised by a 1 transamination reaction, which is catalysed by the GABA transaminase (GABA-T) (Owens and Kriegstein, 2002). Figure 1 Structural formula of GABA 1.2.1 GABAERGIC NEURONS GABA is present in approximately 20-50% of all cerebral cortex synapses (Halasy and Somogyi, 1993; Bloom and Iversen, 1971; Hevers and Lüddens, 1998). In the neocortex, most of the GABA-containing neurons are apparently interneurons with few dendritic spines. They are classified as sparsely spiny, aspiny or smooth cells. These cells are further subdivided into groups such as basket cells, chandelier cells, double bouquet cells, local plexus neurons or neurogliaform cells. GABAergic neurons differ in respect to their morphology, neurochemical composition, somatic location and terminal arborization. GABA containing neurons are apparently distributed throughout the cortical lamina (Owens and Kriegstein, 2002). GABA interneurons are further classified in respect with their intrinsic membrane properties and synaptic connectivity (Connors and Gutnick, 1990; Gupta et al., 2000). Most GABA- containing neurons in the neocortex are generated and migrate from subcortical rather than from cortical locations (Anderson et al., 1997). It has been proposed that in addition to the local circuit neurons, direct GABAergic afferents project to the cortex from the basal forebrain and the zona incerta (Owens and Kriegstein, 2002). 1.2.2 GABAERGIC SYNAPSES Neocortial synapses differ in their ultrastructure and are thus divided into 2 groups: type 1 and type 2 synapses (Gray, 1959; Owens and Kriegstein, 2002).