Concatenation of the Cys-Loop Receptors

CONCATENATION OF THE CYS-LOOP RECEPTORS

GABAA GABAA

ρ αβγ

2 β 2

Master Thesis Miya Marie Kudo Høffding February 2010

Department of Receptor Pharmacology, NeuroSearch A/S Department of Biochemistry, Faculty of Science, University of Copenhagen

Header and footer 1 12 February 2010 Concatenation of the Cys-loop receptors ______

PREFACE

This master thesis is a contribution to the research performed at Deparment of Receptor Pharmacology, NeuroSearch A/S and represents the final part of my education at the Faculty of Science, University of Copenhagen.

The experimental work presented here was carried out at Department of Receptor Pharmacology at NeuroSearch A/S from January 2009 to November 2009. Supervisors were Head of Receptor Pharmacology, Msc. Philip K. Ahring and Research Scientist, Ph.D. Marianne Lerbech Jensen, NeuroSearch A/S, and Associate Professor, Ph.D. Stine Falsig Pedersen, Institute of Molecular Biology & Biochemistry, Department of Biochemistry, University of Copenhagen.

I would like to thank my external supervisors, Philip and Marianne, for giving me the opportunity to take part in the research performed at NeuroSearch A/S. Working in a biopharmaceutical company has been a very valuable experience for me. Further appreciations go to my three supervisors for critically reviewing this thesis and for engaging in numerous interesting scientific discussions. Finally, I am sincerely grateful to my internal supervisor, Stine, for providing immense encouragement and support throughout the project. Your help has been of paramount importance to me, Stine.

I also wish to thank everyone at the Department of Receptor Pharmacology at NeuroSearch, especially Lene G. Larsen and Flemming H. Lausten for technical assistance, and Jeppe K. Christensen for electrophysiological guidance.

Last but not least, my warmest gratitude and love to friends and family.

Copenhagen, 16th of February 2010

Miya Kudo Høffding

Illustration on front page: the homo-oligomeric GABAA ρ receptor (light grey) and the hetero-oligomeric GABAA αβγ receptor (blue and dark grey) with the + and – face of the GABA binding sites marked in red. The GABAA ρ subunits are linked into a penta-concatamer, whereas the α, β, γ subunits are linked into a β-α dimer and a γ-β-α trimer. The linker is in dotted red. The pore-forming M2 segment of each subunit is in dark red.

Concatenation of the Cys-loop receptors ______

TABLE OF CONTENTS

ABSTRACT i DANISH RESUMÉ ii ABBREVIATIONS iii TERMS v FIGURES & TABLES vi

1. INTRODUCTION ...... 1

1.1. THE CYS-LOOP RECEPTORS 1.1.1 Physiological role...... 1 1.1.2 Structure ...... 2 1.1.3 Family members...... 3 1.1.4 Stoichiometry ...... 3 A. nAChRs ...... 3

B. 5-HT3Rs ...... 4

C. GABAARs ...... 4 D. GlyRs ...... 5 1.1.5 Conformational states ...... 5 1.1.6 Functional domains ...... 7 A. Assembly and trafficking...... 7 B. Ligand binding ...... 10 C. Conductance and gating...... 13 D. Ion selectivity ...... 15

1.1.7 GABAA receptors ...... 18

A. Hetero-oligomeric GABAA receptors ...... 18

B. Homo-oligomeric GABAA receptors ...... 19

1.2. CONCATENATION 1.2.1 Applications ...... 23 1.2.2 Concatenation studies of Cys-loop receptors ...... 24 A. Stoichiomety ...... 24 B. Functional role of subunits ...... 25 C. Positional effects of mutations ...... 26 1.2.3 Linker design ...... 27 1.2.4 Precautions in the use of concatenation ...... 30

2. AIM ...... 31

3. EXPERIMENTAL PROCEDURES ...... 32

Concatenation of the Cys-loop receptors ______

3.1. MOLECULAR BIOLOGY 3.1.1 Subunits ...... 33 3.1.2 Concatamers ...... 33

A. GABAA ρ1 ...... 33

B. GABAA α1β3γ2 ...... 34 3.1.3 Overlap Extension Polymerase Chain Reaction ...... 34 3.1.4 Purification of PCR products ...... 38 3.1.5 Agarose gel electrophoresis ...... 39 3.1.6 Determination of DNA concentration ...... 39 3.1.7 TOPO cloning ...... 39 3.1.8 Isolation of plasmid DNA from bacterial colonies ...... 40 3.1.9 Restriction enzymes ...... 41 3.1.10 Ethanol precipitation ...... 41 3.1.11 Sequencing ...... 42 3.1.12 Retransformation ...... 42 3.1.13 Ligation ...... 42 3.1.14 Purification of ligation products ...... 44 3.1.15 cRNA synthesis and purification ...... 46

3.2. CELL CULTURE AND TRANSFECTION 3.2.1 Chinese Hamster Ovary Cells ...... 46 3.2.2 Transient transfections ...... 47 3.2.3 Xenopus laevis oocytes ...... 48 3.2.4 Defolliculation ...... 48 3.2.5 Microinjection ...... 48

3.3. ELECTROPHYSIOLOGY 3.3.1 The patch clamp technique ...... 49 3.3.2 Voltage clamp ...... 50 3.3.3 Errors of voltage clamp ...... 50 A. Series resistance and cell capacitance ...... 50 B. Junction potentials ...... 52 3.3.4 Patch clamp setup for CHO cell recordings ...... 53 3.3.5 Patch clamp setup for Xenopus oocyte recordings ...... 54 3.3.6 Data analysis ...... 55

4. RESULTS ...... 55

4.1. GABAA ρ1 CONCATAMERS

4.1.1 The GABAA ρ1 wildtype receptor ...... 56 4.1.2 Linker optimization ...... 58

4.1.3 Molecular construction of the GABAA ρ1 penta-concatamer with L6ags ...... 61

4.1.4 Molecular construction of the GABAA ρ1 penta-concatamer with L30 ...... 66

4.1.5 Electrophysiological analysis of GABAA ρ1 concatenated constructs ...... 67

Concatenation of the Cys-loop receptors ______

4.2. GABAA α1β3γ2 CONCATAMERS 4.2.1 Maximal current amplitudes ...... 70 4.2.2 GABA concentration-response measurements ...... 71 4.2.3 Diazepam concentration-response measurements ...... 74

5. DISCUSSION ...... 77

5.1. GABAA ρ1 CONCATAMERS 5.1.1 Linker optimization ...... 77 A. Comparison of linker lengths ...... 77 B. Potential formation of loop-over structures ...... 79 C. Potential lack of C-terminal protrusion ...... 81 D. Potential degradation of linker...... 81

5.1.2 GABAA ρ1 penta-concatenation ...... 82 A. Low functional expression of higher-order concatamers ...... 82 B. Potential disruption of the assembly pathway ...... 83 C. Choice of expression system ...... 83 5.1.3 In summary ...... 84

5.2. GABAA α1β3γ2 CONCATAMERS 5.2.1 Functional expression ...... 84 5.2.2 Incomplete incorporation of subunits ...... 85 5.2.3 Re-orientation of dimers ...... 87 5.2.4 In summary ...... 88

6. CONCLUSION ...... 90

7. FUTURE DIRECTIONS ...... 91

8. REFERENCES ...... 92

APPENDICES

A. MEMBRANE POTENTIAL AND CURRENT 102 B. PLASMIDS 104 C. PROTOCOLS 110 D. DRUGS & SOLUTIONS 116

Concatenation of the Cys-loop receptors ______

ABSTRACT

The Cysteine loop receptors are neutrotransmitter-gated ion channels which mediate fast synaptic transmission in the central nervous system (CNS). They are potential therapeutic targets for a broad range of CNS disorders such as anxiety, depression, schizophrenia, Alzheimer’s- and Parkinson’s disease. Therefore, much research has been dedicated to the structural, physiological and pharmacological characterization of these pentameric receptors. One approach involves the concatenation technique, i.e. covalent linkage of subunits on the DNA level to produce receptors of defined arrangement and stoichiometry. This technique enables the selective manipulation of a single subunit that may occur in multiple copies in a receptor, thus elucidating the positional effects of mutations.

In current project concatenation was applied to create a model system for the homo-oligomeric Cysteine loop receptors. The model system comprised a penta-concatamer of five identical linked GABAA ρ1 subunits, which could be distinguished from each other only by the presence of two unique restriction sites within each subunit. This construct would, if functional, be site-specifically modified to reveal potential asymmetries in the contributions of identical subunits of a receptor to agonist binding.

Initially, an experiment was conducted to identify an optimal linker for the penta-concatenation of ρ1 subunits.

Three linkers tethering ρ1 dimers and trimers (L20, L23, L25), and three linkers tethering only dimers (L30, L40,

L6ags) were tested for functional expression in CHO cells by whole-cell voltage clamp. L20, L23 and L25 impaired the functional expression of ρ1, both when dimers and trimers were expressed separately and in pairs, whereas dimers linked with L30, L40, L6ags displayed positive GABA responses in a slightly larger proportion of the cells. A GABAA ρ1 penta-concatamer was successfully synthesized with the shortest of these three linkers, L6ags. Unfortunately, the penta-concatamer produced vanishing little current in CHO cells, HEK cells and Xenopus oocytes. Therefore, no further experiments were conducted with this construct.

A second aim of the project was to electrophysiologically characterize hetero-oligomeric GABAA α1β3γ2 concatamers in Xenopus oocytes and to evaluate the reliability of this concatenation system. Maximal current amplitudes and sensitivity towards GABA and diazepam of the concatenated constructs indicated that β3-α1 dimers and γ2-β3-α1 trimers were able to generate GABA-evoked currents upon separate expression, although to a minor degree compared to the co-expression of these constructs. Furthermore, the results implied that the γ subunit was not incorporated into a subset of the receptors unless significantly over-expressed relative to α and β or covalently linked to these subunits. The dimer + trimer combination appeared to be a successful tool for the generation of wildtype-like GABAA receptors of predefined stoichiometry and arrangement. This was however not the case for the dimer + monomer combination β3-α1 + γ.

Concatenation of the Cys-loop receptors ______RESUMÉ

Cystein loop receptorerne er neurotransmitter-gatede ionkanaler, som medierer hurtig synaptisk transmission i centralnervesystemet (CNS). De udgør potentielle mål for behandlingen af et bredt spektrum af CNS lidelser såsom angst, depression, schizofreni, Alzheimers- og Parkinsons syge. Derfor er megen forskning blevet dedikeret til den strukturelle, fysiologiske og farmakologiske karakterisering af disse pentameriske receptorer. Én tilgang involverer teknikken konkatenering, dvs. kovalent sammenkædning af subunits på DNA niveau for at skabe receptorer af defineret arrangement og støkiometri. Denne teknik muliggør selektiv manipulering af en enkelt subunit, som kan forekomme i flere kopier i en receptor, og belyser således de positionelle effekter af mutationer.

I nærværende projekt blev konkatenering anvendt til at skabe et modelsystem for de homo-oligomeriske Cystein loop receptorer. Modelsystemet bestod af en penta-konkatamer af fem identiske, sammenkædede

GABAA ρ1 subunits, som kun kunne skelnes fra hinanden ved tilstedeværelsen af to unikke restriktionssites i hver subunit. Dette konstrukt ville, hvis funktionelt, blive site-specifikt modificeret for at afsløre potentielle asymmetrier i bidragene fra identiske subunits til agonist binding.

Initielt blev et eksperiment udført for at identificere en optimal linker til penta-konkateneringen af ρ1 subunits.

Tre linkers, der forbandt ρ1 i dimerer og trimerer (L20, L23, L25), og tre linkers, der kun forbandt dimerer (L30,

L40, L6ags) blev testet for funktionel ekspression i CHO celler vha. whole-cell voltage clamp. L20, L23 and L25 forringede den funktionelle expression af ρ1, både når dimerer og trimerer blev udtrykt separat og parvis, mens dimerer forbundede med L30, L40, L6ags udviste positivt GABA respons i en lidt højere andel af cellerne.

En GABAA ρ1 penta-konkatamer blev syntetiseret med den korteste af disse tre linkers, L6ags. Desværre producerede denne penta-konkatamer forsvindende små strømme i CHO celler, HEK celler og i Xenopus oocytter. Derfor blev der ikke udført flere eksperimenter med dette konstrukt.

Et andet formål i projektet var at elektrofysiologisk karakterisere hetero-oligomeriske GABAA α1β3γ2 konkatamerer in Xenopus oocytter og at evaluere pålideligheden af dette konkateneringssystem. De maksimale strømniveauer og GABA- og diazepam sensitiviteten for konkatamerene indikerede, at dimerer og trimerer var i stand til at generere GABA-fremkaldt strøm, når de blev udtrykt separat, dog i ringe grad sammenlignet med co-ekspressionen af disse konstrukter. Resultaterne indikerede ydermere, at γ subuniten ikke inkorporeres i en del af receptorerne, medmindre den er signifikant overudtrykt i forhold til α og β eller kovalent forbundet til disse subunits. Dimer + trimer kombinationen fremstod som et succesfyldt værktøj til genereringen af vildtype-lignende GABAA receptorer af prædefineret støkiometri og arrangement. Dette var dog ikke tilfældet for dimer + monomer kombinationen β3-α1 + γ.

Concatenation of the Cys-loop receptors ______ABBREVIATIONS

AChBP Acetylcholine binding protein

ASIC1A+3R Acid-sensing ion channel type 1A+3 BIG2 Brefeldin A-inhibited GDP/GTP exchange factor 2 BiP Immunoglobulin heavy-chain-binding protein CACA Cis-4-amino-crotonic acid CAMK-II Ca2+/calmodulin dependent kinase II CAMP Cis-2-aminomethyl-cyclopropane carboxylic acid

Cm Membrane capacitance cDNA Complementary/copy deoxyribonucleic acid CHO Chinese hamster ovary CMV Cytomegalovirus CNS Central nervous system COPII Coat protein II cRNA Copy RNA Cys-loop Cysteine loop DEPC Diethyl pyrocarbonate DMEM Dulbecco’s modified eagle medium dNTP Deoxynucleoside triphosphate EC20 Concentration evoking 20% of maximal response EC50 Concentration evoking 50% of maximal response E. coli Escherichia coli EDTA Ethylene diamine tetraacetic acid EGFP Enhanced green fluorescent protein

Emax Concentration evoking maximal response E.R. Endoplasmatic reticulum ERAD E.R. associated degradation FBS Fetal bovine serum GABA γ-amino butyric acid

GABAAR γ-amino butyric acid type A receptor

GABACR γ-amino butyric acid type C receptor GABARAP γ-amino butyric acid receptor-associated-protein GlyR Glycine receptor GODZ Golgi-specific-protein-with-a-DHHC-zinc-finger-domain HAP-1 Huntingtin-associated protein 1 HEK293T Human embryonic kidney cell line 293T

iii

Concatenation of the Cys-loop receptors ______

HEPES N-2-hydroxyetyhlpiperazine-N’-2-ethanesulphonic acid 5-HT 5’ hydroxytryptamine

5-HT3R 5’hydroxytryptamine receptor type 3 I4AA Imidazole-4-acetic acid

Imax Maximal current LB Luria bertani LBA Low Calcium Barth’s solution M1-4 Transmembrane segments 1-4 MBA Modified Calcium Barth’s solution MES 2-N-Morholino ethanesulfonic acid nAChR Nicotinic acetylcholine receptor PAGE Polyacrylamide gel electrophoresis PBS Phosphate-buffered saline PCR Polymerase chain reaction PDI Protein disulfide isomerise pEC50 The negative logarithm of the concentration evoking half maximal response 4-PIOL 5-(4-piperidyl)isoxazol-3-ol PKA Protein Kinase A PKB Protein Kinase B PKC Protein Kinase C PLIC-1 Protein-linking-IAP-to-the-cytoskeleton P4S Piperidine-4-sulfonate RE Restriction enzyme RNA Ribonucleic acid

Rs Series resistance SAP Shrimp alkaline phosphatise SCAM Substituted Cysteine Accessibility Method SDS Sodium dodecyl sulphate S.E.M. Standard error of the mean SEVC Single-electrode voltage clamp Sf-9 Spodoptera frugiperda insect cell line 9 Src Sarcoma tyrosine kinase TACA Trans-4-aminocrotonic acid TAMP Trans-2-aminomethyl-cyclopropane carboxylic acid TE Tris/EDTA buffer TEVC Two-electrode voltage clamp (3α5α)THDOC (3α,5α)-3,21-dihydroxypregnan-20-one THIP 4,5,6,7-tetrahydroisoxazolopyridin-3-ol

Concatenation of the Cys-loop receptors ______

TPMPA (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid TBE Tris/Borate/EDTA buffer tRNA Transfer RNA UV Ultra violet XO Xenopus laevis oocytes ZAC Zinc activated ion channel

TERMS

A brief explanation of common terms applied when describing/discussing concatenation is presented below.

“Actual linker length” Term used by Baumann et al. (2001) to describe the length of the C- terminal protrusion of the first subunit in a tandem, the synthetic linker and the N-terminal of the second subunit in a tandem relative to that of the GABAA β2 subunit. (section 5.1.1A-B)

C-terminal protrusion The C-terminal extracellular tail of a Cys-loop subunit (section 5.1.1)

Dimer / tandem / dual construct Two subunits that are covalently linked to each other

Dimer rearrangement Hypothetical arrangement in which the two subunits of a dimer rotate around each other

Dipentamer Two covalently-linked pentamers formed by five dimmers

Higher-order concatamer “Order” refers to the number of linked subunits. A higher-order concatamer contains more linked subunits than a lower-order concatamer.

Linked / tethered / concatenated Covalently fused from C’ to N’ by a synthetic amino acid sequence

Linker Synthetic amino acid sequence connecting the C-terminus of one subunit to the N-terminus of another subunit in a concatamer

Linker construct PCR fragment containing from 5’ to 3’ the C-terminal part of the first subunit in a tandem followed by the linker sequence, or the linker sequence followed by the N-terminal part of the second subunit in a tandem.

Linker region PCR fragment formed by the overlap extension of two complementary linker constructs. Contains the C-terminal sequence of the first subunit followed by the linker and the N-terminal sequence of the second subunit in tandem.

Loop-over structure (interspersing Hypothetical arrangement in which two subunits that are covalently linked are not positioned adjacent to each other in the receptor subunit)

Concatenation of the Cys-loop receptors ______

Monomeric / free / untethered subunit A single subunit which is not covalently linked to other subunits

N-terminal protrusion The extracellular domain of a Cys-loop subunit excluding the signal peptide (section 5.1.1)

Pentamer / penta-concatamer Five subunits that are covalently linked to each other

Pseudo-hexamer A pentamer receptor formed by three dimers or two trimers with an excessive subunit sticking out of the complex

Tetramer Four subunits that are covalently linked to each other

Trimer / triple construct Three subunits that are covalently linked to each other

Wildtype receptor Receptor composed of five monomers (non-linked subunits)

FIGURES & TABLES

Fig. 1.1. Schematic Cys-loop receptor structure 2 Fig. 1.2. Molecular model of the Cys-loop receptors 5 Fig. 1.3. Model of the conformational states of Cys-loop receptors 6 Fig. 1.4. Intracellular trafficking of GABA-A receptors 9

Fig. 1.5. The GABAA β(+)/(-)α ligand.binding interface 11 Fig. 1.6. The extracellular binding loops A-F of adjacent Cys-loop subunits depicted in side-view 11 Fig. 1.7. Ribbon diagram of a Torpedo nAChR α subunit 11 Fig. 1.8. The gates of Cys-loop receptors 14 Fig. 1.9. The anionic rings contributing to the selectivity of cationic Cys-loop receptors 17 Fig. 1.10. Alignment of the M1-M2 loops and the M2 domains of selected Cys-loop subunits 17

Fig. 1.11. Schematic representation of A: a GABAA αβγ receptor and B: a GABAA ρ receptor 21 Fig. 1.12. The principle of concatenation 23

Fig. 3.1. Primary overlap extension PCR reaction exemplified with templates ρ1A and B 36

Fig. 3.2. Secondary overlap extension PCR reaction exemplified with templates [AC-L6ags] and 37 [L6ags-BN] Fig. 3.3. Flowchart outlining the concatenation strategy for the synthesis of GABAA ρ1 L6ags 45 concatamers Fig. 3.4. Flowchart depicting five different patch clamp configurations 51

Fig. 4.1. Functional expression of GABAA ρ1 receptors in CHO cells 56

Fig. 4.2. GABA and acid sensitivity of CHO cells transfected with GABAA ρ1R or ASIC1A+3R 57

Fig. 4.3. Maximal current amplitudes of GABAA ρ1 concatenated dimers and trimers in CHO cells 58

Concatenation of the Cys-loop receptors ______

Fig. 4.4. Current distribution of CHO cells transfected with L20, L23, L25, L30, L40 and L6ags 60 concatamers Fig. 4.5. Synthesis of linker region 61 Fig. 4.6. Insertion of linker region deletes a XhoI site between the subunits 62 Fig. 4.7. Insertion of linker region creates a SacII site between the subunits 62

Fig. 4.8. Restriction enzyme verification of L6ags dimer, trimer and tetramer 64

Fig. 4.9. Restriction enzyme verification of L6ags pentamer 65

Fig. 4.10. Plasmid illustration of the GABAA ρ1 L6ags penta-concatamer A-B-C-E-D 66

Fig. 4.11. Maximal current amplitudes of L6ags concatamers in CHO cells 67

Fig. 4.12. Current distribution of CHO cells transfected with GABAA ρ1 L6ags concatamers 68

Fig. 4.13. Functional expression of GABAA ρ1 L6ags A-B-C-E-D in CHO cells 69

Fig. 4.14. Maximal current amplitudes of GABAA α1β3 and α1β3γ2 wildtype receptors and 70 concatamers Fig. 4.15. GABA-evoked current responses of GABAA α1β3γ2 concatamers expressed in Xenopus 72 oocytes Fig. 4.16. Potency of GABA on GABAA α1β3γ2 concatamers and wildtype receptors 73

Fig. 4.17. Diazepam modulation of GABAA α1β3γ2 wildtype and concatamer receptors 75 Fig. 5.1. Illustration of the terms used for the comparison of total linker lengths in table 5.1. 79 Fig. 5.2. Unconstrained subunit arrangement due to an excessive linker length 81

Fig. 5.3. Theoretical GABAA α1β3γ2 subunit combinations arising from monomers, dimers and 86 trimers Fig. 5.4. Dimer rearrangement 88

Table 1.1. Overview of Cys-loop family members and cloned subunits 3

Table 1.2. General physiological and pharmacological properties of GABAA receptors 22 Table 1.3. Overview of linkers used in a subset of previous concatenation studies 28

Table 3.1. Overview of unique restriction sites introduced into the GABAA ρ1 subunit isoforms A-E 33

Table 3.2. Previously synthesized GABAA ρ1 concatamers used in the linker optimization experiment 34

Table 3.3. Previously synthesized GABAA α1β3γ2 concatamers 34 Table 3.4. Primers used for overlap extension PCR reactions 35 Table 3.5. Overlap extension PCR reaction step one 36 Table 3.6. Overlap extension PCR reaction: step two 37

Table 3.7. Restriction endonucleases used for the concatenation of GABAA ρ1A-E 41

Table 3.8. Concatenation of ρ1 subunit was performed in two ligation steps for each construct 44 Table 3.9. Plasmid μg ratios for CHO transfection mixtures 47 Table 3.10. cRNA ratios for Xenopus oocyte microinjections 49

Table 4.1. Properties of GABA and diazepam on GABAA α1β3γ2 concatenated dual and triple 76 constructs expressed in Xenopus oocytes

Table 5.1. Comparison of linker optimization experiments 79 Table 5.2 Calculation of the “actual linker length” according to Baumann et al. (2001) 80

vii

Concatenation of the Cys-loop receptors ______1. INTRODUCTION

Throughout the nervous system, neurons communicate at synaptic junctions via electrical or chemical transmission mediated by voltage- or ligand-gated ion channels. The identification and characterization of these channels constitute a major step for understanding the mechanisms underlying almost every physiological function of the brain. Moreover, obtaining detailed knowledge about these ion channels is a prerequisite for improved treatment of various disorders related to the central nervous system (CNS).

This project concerns an ion channel superfamily called the Cysteine loop receptors (Cys-loop receptors). The general structural and physiological properties of these receptors will be outlined below with emphasis on the nicotinic acetylcholine receptors (nAChRs) and the γ-aminobutyric acid type A receptors (GABAARs) which have been most extensively examined in previous studies. Next will be a brief introduction to the GABAA receptors used specifically in this project, followed by an overview of the concatenation technique which provided the major basis of the experimental work performed here. ______

1.1 THE CYSTEINE-LOOP RECEPTORS

1.1.1 Physiological role The Cys-loop receptors are ligand gated ion channels which mediate fast synaptic transmission in the CNS. They are transmembrane multisubunit complexes located in the pre- and postsynaptic terminals of neurons. Presynaptically released neurotransmitter binds to these receptors, thereby inducing a conformational change in the channel pores which enables selective transient ion flux to occur over the membrane (White, 2006). The Cys-loop receptors are either cation-conducting or anion-conducting. The cation-conducting receptors respond to neurotransmitter binding by allowing Na+, Ca2+ and K+ ions to diffuse down their electrochemical gradients. Generally, this depolarizes the cell and drives the membrane potential closer to the threshold level of an action potential, thus increasing the likelihood of signal propagation (see appendix A: The Membrane Potential). The anion-conducting receptors allow diffusion of Cl- ions into the cells from the extracellular milieu thus stabilizing or hyperpolarizing the membrane potential and reducing the likelihood of action potential firing (Connolly and Wafford, 2004). In other words, the general role of Cys-loop receptors is to mediate and regulate communication between neurons in the CNS by converting chemical neurotransmitter signals into excitatory or inhibitory synaptic potentials.

Concatenation of the Cys-loop receptors ______

1.1.2 Structure Members of the Cys-loop superfamily share a similar receptor architecture of five subunits assembled pseudosymmetrically around a central ion selective pore. A highly simplified structural representation of a Cys-loop receptor subunit and the pentameric arrangement is shown in figure 1.1. Each subunit comprises a large extracellular N-terminal domain followed by 4 hydrophobic transmembrane segments (M1-M4) with a large intracellular loop connecting segment 3 and 4, and finally a short extracellular C-terminus (Brejc et al., 2001; Noda et al., 1983). As will be described further in the following text, the N-terminal domain is responsible for agonist binding and subunit assembly, whereas the intracellular M1-M2 linker and the M2 segment are important for ion selectivity and conductance. The short extracellular loop connecting M2 and M3 has been shown to contribute to the coupling of agonist binding to channel opening, and the large intracellular M3-M4 loop interacts with various intracellular proteins and is important for subunit trafficking to the cell surface. The N-terminal extracellular domain contains a characteristic loop structure created by the bridging of two conserved cysteines separated by 13 amino acids. This loop is a feature of all subunits, hence the name: Cys-loop receptors (Kao and Karlin, 1986; Schofield et al., 1987). The functional role of the cys- loop will be outlined in section 1.1.6C.

Fig. 1.1. Schematic Cys-loop A Ligand binding, B receptor structure. A: Pentameric assembly Cys-loop arrangement of subunits around the Gating central ion conducting pore viewed COOH Extracellular from the extracellular face. The red segment represents M2. B.

M1 M2 M3 M4 Schematic representation of a Cys- loop receptor subunit showing the large extracellular domain, the four Ion selectivity, Intracellular transmembrane segments and the conductance large cytoplasmic loop between M3 and M4. The cys-loop is indicated Interaction with intracellular proteins, by the arrow. The position is subcellular distribution symbolic. Both figures are modified from Jensen (2003).

A Cys-loop receptor subunit has a molecular weight of approximately 50 kD and constitutes a single polypeptide chain of 400-500 amino acid residues. A signal peptide of 20-30 amino residues is located at the N-terminal end, followed by a large extracellular domain of 200-250 3residues. Each of the four membrane- spanning segments is composed of approximately 20-30 residues, and the large cytoplasmic loop between M3 and M4 contains approximately 90-150 residues. The M1-M2 loop, the M2-M3 loop and the C-terminal protrusion are generally composed of less than 30 residues each (UniprotKB, 2009). The dimensions of a subunit have been estimated to 30 x 40 x 160 Å with the long axis perpendicular to the membrane plane (Unwin, 2005).

Concatenation of the Cys-loop receptors ______

1.1.3 Family members The Cys-loop receptors comprise five families of ligand-gated ion channels: the cation selective nicotinic acetylcholine receptors (nAChR) and 5-hydroxytryptamine type three receptors (5-HT3R) which respond to the excitatory neurotransmitters, acetylcholine and 5-HT, respectively, and the anion selective γ-aminobutyric acid type A receptors (GABAAR) and glycine receptors (glyR) which respond to the inhibitory neurotransmitters, GABA and glycine, respectively. Additionally a zinc activated cation channel (ZAC) has been identified. Very little is known about this receptor, hence it will not be described further (Connolly and Wafford, 2004; Davies et al., 2003).

For each receptor family multiple subunit classes and subtypes have been cloned. These subunits combine to form different receptor subtypes characterized by distinct physiological and pharmacological traits. Table 1.1 presents the subunit repertoire within each family and the stoichiometries of the most common Cys-loop receptors.

Cloned Pathophysiological/therapeutic Family Stoichiometries* References subunits regimen nAChR α1, β1, γ, ε, δ β-α-γ/ε-α-δ Myasthenia gravis (Boulter et al., 1987; α2-6,10, β2-4 α/β-α-β-α-β Alzheimer’s - and Parkinson’s Boulter et al., 1986; disease, schizophrenia, Tourette’s Karlin, 2002; Zouridakis α7-9 α-α-α-α-α syndrome, depression, tobacco et al., 2009) dependence, attention-deficit hyperactivity disorder, neuropathic pain 5-HT3R 5-HT3A-E A-A-A-A-A Emesis, anxiety, drug addiction, (Barnes et al., 2009; B-A-B-A-B chronic neuropathic pain, Davies et al., 1999; fibromyalgia, migraine Faerber et al., 2007; Maricq et al., 1991; Niesler et al., 2007) GABAAR α1-6, β1-3, γ1- γ-β-α-β-α Anxiety, depression, schizophrenia, (D'Hulst et al., 2009; 3, δ, ε, π, θ δ- β-α-β-α epilepsy, insomnia, Huntington’s Macdonald and Olsen, ρ ρ ρ ρ ρ disease 1994; Whiting et al., ρ1-3 1- 1- 1- 1- 1 1999) GlyR α1-4, β α/β-α-β-α-β Peripheral inflammatory pain (Grenningloh et al., 1990; sensation, hyperekplexia Grenningloh et al., 1987; Kuhse et al., 1990; Lynch, 2009; Matzenbach et al., 1994)

Table 1.1. Overview of Cys-loop family members and cloned subunits. *In the counterclockwise arrangement viewed from the extracellular cleft as depicted in position 1-5 in fig. 1.2A

1.1.4 Stoichiometry

A. nAChRs The nAChRs are divided into two groups: those localized at the neuromuscular junction (“muscular”), and those occurring in the central nervous system (“neuronal”). The muscular nAChRs are composed of two α1

Concatenation of the Cys-loop receptors ______subunits, one β1 subunit, one γ or ε subunit and one δ subunit in the arrangement β1-α1-γ/ε-α1-δ when viewed counterclockwise from the synaptic cleft. The γ subunit is found in embryonic nAChRs and in the nAChRs of the electric ray Torpedo marmorata whereas the ε subunit is found in adult muscle nAChRs (Unwin 2005).

The neuronal nAChRs are either homo-oligomeric and composed of α7-9 subunits or hetero-oligomeric and composed of α2-6,10 in complex with β2-4. Metabolic labelling and subunit-specific purification of heterologously expressed α4β2 nAChRs has indicated a receptor composition of two α4 subunits and three β2 subunits (Anand et al., 1991). This result was also obtained in a reporter mutation assay on nAChR stoichiometry conducted by Boorman et al. (2000). However, injection of α4 and β2 cRNA in different ratios into oocytes has revealed two pharmacologically distinct α4β2 receptor subtypes characterized by high and low agonist affinity (Nelson et al., 2003). Based on a reporter mutation assay, the stoichiometries of these two populations were determined to be (α4)2(β2)3 for the high-affinity component and (α4)3(β2)2 for the low-affinity component (Moroni and Bermudez, 2006).

B. 5-HT3Rs

Of the five 5-HT3 subunits cloned, only 5-HT3A is able to form functional homo-oligomeric receptors upon heterologous expression. The naturally occurring 5-HT3Rs are believed to be hetero-oligomeric and composed of primarily 5-HT3A and 5-HT3B subunits. 5-HT3D and 5-HT3E subunits seem to play a modulatory role in the gastro-intestinal tract since their expression is limited to this region predominantly (Niesler et al., 2007).

Recently, atomic force microscopy revealed that hetero-oligomeric 5-HT3Rs were composed of two 5-HT3A subunits and three 5-HT3B subunits in the arrangement: B-A-B-A-B (Barrera et al., 2005).

C. GABAARs

GABAARs are predominantly hetero-oligomeric. Autoradiography, immunolabelling and co- immunoprecipitation studies have revealed that the majority of these receptors are composed of α, β and γ subunits with the subtype α1β2γ2 comprising ~ 60% of the receptors in the brain. α2β3γ2 and α3β3γ2 receptors are expressed to a minor degree of about 10-15 % (Barrera and Edwardson, 2008; Knight et al., 2000). The αβγ receptors are composed of two α subunits, two β subunits and one γ as determined by subunit labelling, incorporation of reporter mutations and Förster resonance energy transfer (Benke et al., 1994; Chang et al., 1996; Farrar et al., 1999; Knight et al., 2000; Tretter et al., 1997). This has also been confirmed by concatenation studies as described in section 1.2.2A, which additionally suggests the counterclockwise arrangement γ-β-α-β-α when viewed from the synaptic cleft (Baumann et al., 2002).

Extrasynaptically located GABAARs containing δ subunits combined with α4/6 and β2/3 have been identified in dendritic and somatic membranes of granule cells in the cerebellum (Nusser et al., 1998). By application of atomic force microscopy the counterclockwise arrangement of α4β3δ receptors was determined to be δ-β-α-β-α when viewed from the synaptic cleft (Barrera et al., 2008). A more promiscuous role of the δ subunit was

Concatenation of the Cys-loop receptors ______observed in an α6β3δ concatenation study described in section 1.2.2B. Here it was indicated that the δ subunit could participate in the formation of an agonist binding site, and was able to occupy multiple positions in a pentamer (Baur et al., 2009).

The less characterized ε and π subunits are believed to replace the γ subunit in a subset of the GABAARs in hypothalamus and hippocampus or in the uterus, respectively (Hedblom and Kirkness, 1997; Whiting et al.,

1997) while θ subunits have been found to co-assemble with α2, β1, and γ1 subunits in the striatum of rats

(Bonnert et al., 1999). GABAA ρ1-3 form homo-oligomeric receptors and are primarily expressed in the retina. They will be described in section 1.1.7B.

D. GlyRs Heterologously expressed GlyRs can form both homo-oligomeric and hetero-oligomeric receptors. In vivo, embryonic GlyRs are primarily composed of α2 subunits solely, whereas the majority of GlyRs in the adult spinal cord assemble into hetero-pentamers of α and β subunits (Laube et al., 2002). Hetero-oligomeric α1β

GlyRs have been proposed to contain three α1 subunits and two β subunits, based on the observation that the effects of a reporter mutation on agonist affinity were greater when the mutation was borne by an α subunit (Burzomato et al., 2003). This finding contradicts earlier results obtained by densitometry measurements of cross-linked GlyRs from rat spinal cord, which indicate a GlyR stoichiometry of two α subunits and three β subunits (Langosch et al., 1988). Newer data generated from quantitative comparison of [35S] methionine- labelled GlyR α1 receptors and α1β receptors purified from whole cells or from the plasma membrane, supports a stoichiometry of (α1)2(β)3 in the arrangement β-α-β-α-β (Grudzinska et al., 2005).

Fig. 1.2. Molecular model of the Cys-loop A B receptors. A. Top view of pentamer corresponding to the schematic representation in fig. 1.1A. Subunits are numbered counterclockwise. Positions 2 and 4 symbolize the principal subunits. B. Side view of pentamer depicting from the top the extracellular domains composed of 10 beta strands and a small alpha helix per subunit and the four transmembrane segments M1- M4 per subunit. The cytoplasmic M3-M4 loop is not shown. The figure is from Ericksen and Boileau (2007).

1.1.5 Conformational states Cys-loop receptors are gated allosterically. This means that agonists bind to the receptors at a site that is structurally segregated from the active site (the pore), and the binding will cause a reversible conformational

Concatenation of the Cys-loop receptors ______

change in the quarternary structure of the receptor which favours channel opening (Changeux and Edelstein, 1998; Connolly and Wafford, 2004). It is believed that Cys-loop receptors cycle between different conformational states. In a highly simplified model three such states have been defined:

the resting state denotes the closed conformation of the receptor channel, which is most stable when no agonist is bound the active state denotes the open, ion conducting conformation of the channel which is transiently promoted when agonist is bound the desensitized state denotes a refractory, non-conducting conformation, which is stabilized upon prolonged agonist binding

The conformation of the resting state and the desensitized state differs although the channel is closed in both cases, and the block of ion conduction in these distinct conformations may be mediated by different gates as depicted in fig. 1.8 (Wilson and Karlin, 2001). At a given concentration of agonist, all three states may be represented in a receptor population, but the relative affinity for these states varies among different receptor families and subtypes.

Jones and Westbrook (1995) demonstrated with paired pulse electrophysiological experiments that GABAA receptors did not immediately return to the resting state upon channel closure, but entered a refractory, agonist-insensitive desensitized state in which the current decay could be fitted with two time constants ranging from a few ms to 10-100 ms. They proposed a model in which desensitization occurs in two stages defined by the number of agonist molecules bound to the receptors. This is depicted in fig. 1.3.

Fig. 1.3. Model of the conformational states of Cys-loop receptors A. Model presenting coupling of the resting state (R) to the monoliganded (B1) and doubly liganded (B2) state

with transition to a brief (O1) or a longer (O2) open state and a slow (Dslow) or fast (Dfast) desensitized state. The model was presented by Jones and Westbrook, (1995) and was modified by Jensen et al.. (2003).

Binding of one molecule (B1) allows the channel to enter a brief open state (O1) and a long desensitized state

(Dslow) whereas binding of two molecules (B2) allows entrance into a longer open state (O2) and a rapidly

equilibrating desensitized state (Dfast). The model assumes that the receptors must bind two agonist molecules to attain the longer open state. However, as will be outlined in section 1.1.6B this is not necessarily the case

for the homo-oligomeric receptors.

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Concatenation of the Cys-loop receptors ______

1.1.6 Functional domains

The Cys-loop receptors can be roughly dissected into different functional domains responsible for assembly and expression of subunits, allosteric ligand binding, gating, ion permeation and charge selectivity. When referring to distinct amino acid positions within these domains, residues will generally be numbered from the N- to C-terminus of a subunit polypeptide as is the case in section 1.1.6A-B. However, a different numbering system is applied when describing residues in the pore of the receptor in order to facilitate comparisons between different Cys-loop family members. In this system the 0’position denotes a conserved positively charged residue at the cytoplasmic end of segment M2. Negative positions represent the direction from 0’ towards the cytoplasmic M1-M2 loop whereas positive positions represent the direction from 0’ towards the extracellular loop connecting segments M2 and M3. This numbering system is illustrated in fig. 1.10 and will be used in sections 1.1.6C-D.

A. Assembly and trafficking The assembly process takes place in the endoplasmatic reticulum (E.R.) where the nascent polypeptide of a Cys-loop subunit is threaded cotranslationally through the E.R. membrane in the direction from the N- terminus to the C terminus (Green, 1999). In the E.R. lumen cleavage of the signal sequence, oxidation of disulfide bonds and N-glycosylation of specific residues are mediated by chaperone proteins such as immunoglobulin heavy-chain-binding protein (BiP), protein disulfide isomerase (PDI) and calnexin (Millar and Harkness, 2008). The entire process of translocation, maturation and assembly is slow ( 2-3 hours), and it has been estimated that less than half of the synthesized subunits are surface-expressed in vivo (Merlie and Lindstrom, 1983).

Oligomerization of Cys-loop receptors in the E.R. involves specific assembly signals which enable subunits to selectively recognize their neighbours in the pentamer (Bollan et al., 2003). These signals have been mapped by the design of chimeras in which sequences of assembly-incompetent subunits were replaced with homologous sequences of assembly-competent subunits or vice versa. The influence of the transferred sequences on oligomerization, if any, was inferred by the presence or absence of interactions between the chimera and wildtype subunits observed in co-immunoprecipitation experiments combined with immunofluorescence localization and electrophysiological assessment of functional expression. By this approach it was shown that the assembly of GABAA receptors is dictated by several subunit-specific assembly motifs within a region spanning from position ~ 52-104 in the N-terminal domain of each subunit (Klausberger et al., 2001; Sarto et al., 2002; Taylor et al., 2000). Likewise, “assembly boxes” important for mutual subunit recognition have been identified in the extracellular domains of αβγδ nAChRs and GlyRs. The positions of these boxes do not appear to be conserved among Cys-loop family members, nor are the same motifs used for the assembly of homo-oligomeric and hetero-oligomeric receptors within a family (Griffon et

Concatenation of the Cys-loop receptors ______al., 1999; Kreienkamp et al., 1995; Yu and Hall, 1991). It is however a general feature of the Cys-loop receptors that the majority of these assembly motifs reside within the N-terminal domain.

The oligomerization pathway of nAChRs has been exploited by the identification of potential assembly intermediates. These were generated by expression of different subunit combinations and studied by pulse- chase experiments, co-immunoprecipitation, surface binding and sucrose gradient sedimentation. The results gave rise to two models, both of which build on the principle that assembly of nAChRs occurs in a stepwise manner with formation of distinct oligomeric subunit intermediates preceeding the final pentameric arrangement (Green, 1999). The heterodimer model states that assembly of αβγδ nAChRs begins with folding of the α subunit into a mature conformation which associates with γ or δ subunits in a parallel fashion to form αγ and αδ heterodimers. These dimers then associate with the β subunit to form functional αβγδ pentamers that are trafficked to the cell surface (Blount et al., 1990; Gu et al., 1991; Saedi et al., 1991). The sequential model suggests a rapid, possibly co-translational association of α, β and γ subunits into trimers prior to α subunit maturation. Following this event, the δ subunit joins to create a tetrameric complex to which the final α subunit is subsequently added. Processing and folding is believed to occur continuously throughout this pathway with each assembly step functioning as a checkpoint to prevent mis-folded or –assembled intermediates from participating in later oligomerization reactions (Green, 1999; Green and Wanamaker, 1997). It is currently unknown whether other Cys-loop receptors assemble by such mechanisms. For the hetero-oligomeric GABAA receptors it has been shown that in heterologous expression systems, co- expression of α and β subunits is required for access of the receptors to the cell surface. Combining γ2 with only α1 or β2 causes E.R. retention, as does the separate expression of these subunits (Connolly et al., 1996).

Upon correct oligomerization, receptors are transported to the Golgi apparatus via COPII coated vesicles. Presumably, this requires both masking of E.R. retention signals and exposure of E.R. export signals in correctly folded and oligomerized receptors (Ren et al., 2005). Several such trafficking motifs have been identified in the large cytoplasmic loop between transmembrane segments three and four (Griffon et al., 1999; Keller et al., 2001; Lo et al., 2008; Ren et al., 2005). Cytoplasmic E.R. retrieval signals have also been identified on the short intracellular loop between M1 and M2 of 5-HT3ARs and GABAAR γ subunits. These signals return proteins from the cis-Golgi department to the E.R. unless masked by correct folding and co- assembly with other subunits (Connolly, 2008).

Transport of Cys-loop receptors through the secretory pathway is facilitated by a range of proteins associated with the E.R. and Golgi membranes. For hetero-oligomeric GABAA receptors these proteins include the

GABA-receptor-associated-protein (GABARAP), which interacts with the intracellular M3-M4 loop of γ2 subunits; the protein-linking-IAP-to-the-cytoskeleton (PLIC-1) which interacts with the intracellular M3-M4 loop of α1-3,6 and β1-3 subunits to protect the receptors from proteasome-mediated degradation, and the Golgi-

Concatenation of the Cys-loop receptors ______specific-protein-with-a-DHHC-zinc-finger-domain (GODZ), which palmitoylates the γ2 subunit to stabilize surface clustering of receptors (Connolly, 2008; Saliba et al., 2008). Furthermore, the Brefeldin A-inhibited

GDP/GTP exchange factor 2 (BIG2) binds to the M3-M4 loop of GABAA β subunits and assists the exocytic transport of receptors through the Trans-Golgi Network. At the plasma membrane the scaffold protein gephyrin stabilizes the receptors by coupling to the actin cytoskeleton (Tretter and Moss, 2008). The transport proteins are depicted in fig. 1.4.

Fig. 1.4. Intracellular trafficking of GABAA receptors. Correctly folded and assembled receptors are transported from the E.R. via the Golgi apparatus to the plasma membrane in the secretory pathway. This transport is assisted by several trafficking proteins. In the membrane, receptors move laterally to their synaptic or extrasynaptic destinations. Endocytosis via clathrin-coated pits is regulated by phosphorylation. Receptors are degraded in the lysosomes after retrieval from the plasma membrane or by E.R. associated degradation (ERAD). The figure is from Tretter and Moss (2008).

The amount of surface-expressed receptors is regulated by constitutive dynamin-dependent endocytosis via clathrin-coated pits. The adaptor complex AP-2 recognizes binding motifs in the cytoplasmic M3-M4 loops of subunits and facilitates the internalization of receptors. For the GABAA receptors a significant proportion of the receptors are rapidly recycled to the plasma membrane. The Huntingtin-associated protein 1 (HAP1) has been proposed to facilitate this process as depicted in fig. 1.4 (Tretter and Moss, 2008). The remaining

Concatenation of the Cys-loop receptors ______receptors are subjected to degradation in the lysosomes. Endocytosis is in turn regulated by kinases such as the protein kinases A-C (PKA, PKB, PKC), the Ca2+/calmodulin dependent kinase II (CaMK-II) and the Sarcoma tyrosine kinases (Src). Tuning of the level of GABAergic inhibition also appears to be regulated by polyubiquitination which targets newly translated or assembled subunits in the E.R. to the proteasome for degradation in response to blockade of neuronal activity (Saliba et al., 2007). This process is known as E.R. associated degradation (ERAD) (Tretter and Moss, 2008).

Overall, it is evident that the folding, assembly and trafficking of Cys-loop receptors to and from the plasma membrane occurs by stringently controlled complex pathways involving multiple conformational changes and interaction of subunits with each other and with chaperones and transport proteins. This ensures the selective expression of functional receptors on the cell surface.

B. Ligand binding Structural studies of the ligand binding domains of Cys-loop receptors have been greatly enhanced by crystallization of the Acetylcholine-binding protein (AChBP) from the glial cells of the mollusca Lymnaea stagnalis, Aplysia californica and Bulinus truncatus (Rucktooa et al., 2009) . The soluble protein forms a homo-pentamer which is structurally homologous to the extracellular domain of nAChR α subunits. It contains the majority of conserved nAChR residues and binds known nAChR agonists and competitive antagonists with affinities resembling those of neuronal α7/9 receptors. Therefore its high resolution structure (down to 1.75 Å) has been used as a model system for the ligand-binding domain of Cys-loop receptors (Brejc et al., 2001; Karlin, 2002).

The agonist binding sites of Cys-loop receptors are extracellular pocket-like structures located in the N- terminal domains at the interface of two neighbouring subunits (Corringer et al., 2000). Each binding site is mainly composed of β sheets connected by a series of flexible loop structures donated from the principal face of one subunit and from the complementary face of an adjacent subunit. These extracellular faces are denoted + and – when viewed counterclockwise and clockwise, respectively (Sine et al., 1995). This is shown in fig. 1.2A.

Mutagenesis and photoaffinity labelling studies of hetero-oligomeric muscle nAChRs combined with knowledge from the AChBP structure suggest that the principle part of a binding pocket is formed by three clusters of binding residues designated loops A-C. These loops reside between β strands on the + side of a nAChR α1 subunit and project aromatic residues into the subunit interface. The complementary part is formed by three loops D-F residing within β strands conferred to the – side of either a δ or γ subunit. Loops D-F project negatively charged, hydrophobic and aromatic residues into the binding site. (Brejc et al., 2001;

Concatenation of the Cys-loop receptors ______

Czajkowski and Karlin, 1995; Karlin, 2002; Sine et al., 1995). The relative positions of these binding loops are depicted in fig. 1.6.

Fig. 1.5. The GABAA β(+)/(-)α binding interface. Regions involved in agonist binding are shown in yellow. A GABA molecule is shown in CPK (Corey–Pauling–Koltun) format. The pore forming M2 segment is shown in red. Regions involved in gating are in orange. The M3-M4 loop is

reduced. The figure is from Kash et al.. (2004).

Fig. 1.7. Ribbon diagram of a Torpedo nAChR α subunit. The structure is based on 4Å resolution electron images of tubular Torpedo membranes. The subunit is depicted parallel to the membrane plane. The extracellular domain is Fig. 1.6. The extracellular binding loops A-F of adjacent composed of 10 β-strands which are shown in red and blue.The transmembrane segments M1-M4 are in yellow, as Cys-loop subunits depicted in side-view. A (red), B (light is the cytoplasmic loop connecting M3 and M4. Part of this blue) and C (yellow) reside on the principal subunit. D loop is missing in this figure. The loop structures which (green), E (orange) and F (dark blue) reside on the contribute to gating are marked with red boxes. Figure from complementary subunit. The figure is from Thompson and Unwin (2005). Lummis (2006). 11

Concatenation of the Cys-loop receptors ______

The binding sites of hetero-oligomeric GABAA receptors are formed at the interface between the – side of α subunits and the + side of β subunits as shown in fig. 1.5. In fig. 1.2A this corresponds to the interface between subunits 2 + 3, and 4 + 5 in a 1-5 arrangement of γ-β-α-β-α. Application of the Substituted Cysteine Accessibility Method (SCAM) combined with electrophysiology and homology modelling of the AChBP has revealed that the binding sites of hetero-oligomeric GABAA receptors resemble those of the AChBP and nAChRs with A-F loop structures created by distinct clusters of pocket lining residues on the – and + face of α and β subunits, respectively (Boileau et al., 2002; Holden and Czajkowski, 2002). By the same approach it has been shown that the ligand binding pocket is shaped as a deep narrow cleft which appears to constrict upon activation. Binding of an antagonist larger than GABA is likely to impede this constriction and prevent gating of the channel (Wagner and Czajkowski, 2001).

Homology modelling of the 5-HT3R and GlyR extracellular domains has also been performed using on the structurally related AChBP as template. For the 5-HT3Rs natural-, unnatural- and scanning alanine mutagenesis studies identified clusters of binding site residues distributed in loops corresponding to A-E of

AChBP at the interface of two 5-HT3A subunits. Cation-π interactions as well as aromatic contacts to agonist molecules were found at positions equivalent to key binding residues in other Cys-loop receptors (Thompson and Lummis, 2006). This was also the case for the homo- and hetero-oligomeric GlyRs, indicating a conserved ligand-binding mechanism among members of the Cys-loop family (Grudzinska et al. 2005).

Hetero-oligomeric Cys-loop receptors are believed to contain two agonist binding sites, each of which must be occupied by an agonist molecule for complete activation (Baumann et al., 2003; Karlin, 2002). The homo-oligomeric receptors are composed of five identical subunits, hence each subunit is able to form both the principal and complementary face of a binding site yielding a receptor with five binding sites. However, mutational studies of homo-oligomeric Cys-loop receptors combined with electrophysiological assessment of functional expression have in several cases revealed that these receptors can be fully activated with less than five intact binding sites. In 1996 Amin and Weiss created chimaeric GABAA ρ1 receptors containing a reduced number of binding sites to determine the number of agonist molecules required to activate the receptor. They co-expressed wildtype and activation-impaired ρ1 subunits in different ratios in Xenopus oocytes and compared the concentration-response curve of each subunit mixture to concentration- response relationships predicted by a binomial relation for subunit assembly. The results indicated that three out of five equivalent and independent agonist binding sites should be occupied by agonist to gate the ion permeable pore. Amin and Weiss suggested that the purpose of the two additional binding sites could be to increase the probability of agonist binding and to stabilize the open conformation of the receptor (Amin and

Weiss, 1996). Recently, Bouzat and group also observed that chimeras of the homo-oligomeric nAChR α7 extracellular domain and the 5-HT3A transmembrane and cytoplasmic region could be activated with less than

Concatenation of the Cys-loop receptors ______five intact binding sites (Rayes et al., 2009). They introduced a binding mutation into subunits that additionally contained a reporter mutation which altered the single-channel current amplitude. By combining the mutant and non-mutant chimeras in different ratios and observing the single-channel current amplitude, the number of intact binding sites in each receptor which elicited channel openings could be deduced. They found that two non-consecutive binding sites could be disabled without affecting the maximal open channel lifetime of receptors. Furthermore, macroscopic current recordings indicated that the presence of at least two nonconsecutive binding sites was required for rapid activation and steady-state desensitization of the receptors (Rayes et al., 2009). Therefore it was concluded that the agonist ligation of two nonconsecutive binding sites was sufficient to produce burst openings, whereas ligation of three intact binding sites at nonconsecutive subunit interfaces was required to maximize the stability of the open channel. Such mechanism in which occupation of a third subunit interface potentiates burst openings induced by two agonist binding sites corresponds to the modulation of GABA-evoked currents by benzodiazepines (described in section 1.1.7A) (Rayes et al., 2009). Activation mechanisms involving three agonist binding sites have also been suggested for the homo-oligomeric α1 GlyRs (Beato et al., 2002, 2004; Gentet and Clements, 2002) and for the 5-HT3A receptor (Mott et al., 2001). Molecular dynamics simulation of a nAChR α7 homology model has led to the suggestion that asymmetric motions of the five identical α7 subunits in a receptor may cause a variable binding strength at different interfaces of the subunits. The simulation proposes that the interfaces between subunits 1+2 and 4+5 in a homo-oligomeric receptor corresponding to fig. 1.2A might bind the agonist more readily and thereby induce a more symmetrical arrangement of the subunits which improves the subsequent binding of agonist at the remaining interfaces (Henchman et al., 2003).

In summary, it appears as if the homo-oligomeric Cys-loop receptors can be activated with less than five agonist-occupied binding sites and that some of these sites serve to stabilize the open conformation of the receptor channel rather than increasing the probability of opening. Likely the binding of agonist to these receptors occurs in a position-selective manner which is perhaps dictated by small asymmetrical movements of the five identical subunits in the receptor complex.

C. Conductance and gating The channel pores of Cys-loop receptors provide a hydrated environment which upon gating allows selected ions to traverse the plasma membrane in the direction dictated by their electrochemical gradients. These pores are shaped by five M2 segments, one donated from each subunit (Akabas et al., 1994; Imoto et al., 1988; Imoto et al., 1986; Leonard et al., 1988). The other three transmembrane segments, M1, M3, and M4 form an outer ring which shields M2 from the lipid bilayer (Miyazawa et al., 2003). This is shown in fig. 1.1A. SCAM studies proposed an α helical structure of the pore lining domain due to water-accessibility of every third amino acid residue in M2 segment (Akabas et al., 1994; Reeves et al., 2001). This finding was confirmed by

Concatenation of the Cys-loop receptors ______the Unwin team in a series of electron microscopy analyses of crystalline Torpedo ray post synaptic membranes (Miyazawa et al., 2003; Unwin, 1995, 1998, 2005). By freeze-trapping Torpedo nAChRs in open, closed and desensitized states, structural differences associated with the activation of these receptors could be detected down to 4Å resolution. Images of the resting state depicted five M2 alpha helices which tilted radially and symmetrically inwards with bends near the middle plane of the membrane facing toward the axis of the pore. Side-to-side interactions between bulky, hydrophobic side chains of homologous residues in this bent region seemed to create a tight constriction, a so-called “hydrophobic girdle”, with a minimum radial distance from the central axis to the nearest surface of ~3Å. This constriction was predicted to be tight enough to prevent the permeation of sodium or potassium ions in their hydrated states. Therefore, it was nominated as the gate of the receptor (Miyazawa et al., 2003). In accordance with this, a centrally located channel gate was identified in a SCAM study of 5-HT3AR and in a blocker-trapping experiment of GABAAR with picrotoxin

(Bali and Akabas, 2007; Panicker et al., 2002). However, SCAM studies of muscle nAChRs and α1β2γ2

GABAARs inferred that the gate of a closed pore should be positioned more intracellularly near the cytoplasmic end of the membrane traversing region (Wilson and Karlin, 1998; Xu and Akabas, 1993, 1996). Additionally, it was postulated that the block of ion permeation in the resting and desensitized state of nAChRs was mediated by different gates (Wilson and Karlin, 2001). The positions of these gates are depicted in fig. 1.8.

Fig. 1.8. The gates of Cys-loop receptors. Resting Open Desensitized

20’ 20’ 20’ The model depicts two pore-lining M2 segments 16’ 16’ 16’ of the nAChR with individual amino acid 13’ 13’ 13’ residues represented by circles. The blue circles

9’ 9’ 9’ represent residues which have been analyzed by

6’ SCAM. The gates of the resting and desensitized 6’ 6’

2’ states are shown in red. The figure is from Wilson 2’ 2’ and Karlin (2001) and was modified by Jensen et -1’ -1’ -1’ al.. (2003). -5’ -5’ -5’

Recently, time-resolved photolabeling of Torpedo nAChRs in the fast and slow desensitized state showed decreased labeling of the channel lumen at a highly conserved 9’-leucine residue of the M2 segment during transition from the resting to open and open to fast desensitized state. The position of this residue within the channel pore can be seen in fig. 1.8 and 1.10. The result indicates that the conformation of this central region of the pore changes with each state of the receptors, supporting the hypothesis of different gates for channel opening and desensitization (Yamodo et al., 2009).

Concatenation of the Cys-loop receptors ______

Based on a wide array of mutational, computational and electrophysiological studies a unified gating mechanism of Cys-loop receptors is emerging (Kash et al., 2003; Lee et al., 2009; Lee and Sine, 2005; Lynch et al., 2001; Unwin, 2005). The coupling of ligand binding to channel opening is believed to involve a concerted motion of at least three prominent loop structures of the extracellular domain. These loops are boxed in red in fig. 1.7. One of the loops is the C loop situated at the binding site interface on the principal subunit. The C loop connects β strand 9 with β strand 10, which forms the pre-M1 region between the ligand binding domain and the transmembrane domain. The two other loops reside between β strands 6 and 7 (cys- loop), and between β strands one and two (loop 2), respectively. These loops face the membrane and project into a cavity framed by the pre-M1 region and the short extracellular linker connecting transmembrane segments M2 and M3. Upon docking of agonist, the C loop is proposed to change from an open to a closed conformation which caps the binding pocket and traps the agonist within the binding site. This capping motion projects through the rigid body of β strand 10 to the pre-M1 region where residues interact with loop 2 and the cys-loop. At the same time these loops also interact with residues of the short extracellular linker connecting the transmembrane segments M2 and M3. In this way, the cys-loop and loop 2 seem to functionally couple movements in the extracellular pre-M1 region to the transmembrane domain and transduce agonist binding into channel gating (Lee et al., 2009). The regions involved in this mechanism are marked in orange in fig. 1.5. According to the cryo-electron microscopical studies conducted by Unwin and group the allosteric movement of the extracellular domains of particularly the α subunits of nAChRs is converted to a rotating motion of the inner sheets of the pore. The rotation turns the central bends of the α helices over to the side in a right-handed twisted barrel configuration thus weakening the hydrophobic interactions of the girdle and widening the channel lumen in the middle of the pore (Unwin, 1995). In a different model it has been proposed that the pathway from agonist binding to channel opening involves an outward movement of the F loop (dark blue in fig. 1.6) and the cys-loop toward the C loop which may additionally cause an outward movement of the pre-M1 region and the M1 segment. Together these outward movements couple to the M2–M3 linker directly or via loop 2, pulling M2 and M3 into a pore dilating conformation with the segments tilting outward as a rigid body parallel to the membrane (Chang et al., 2009).

D. Ion selectivity The ion selectivity of Cys-loop receptors is based on rings of charged or polar residues situated at equivalent positions in the M1-M2 loop region and the M2 segment of each of the four subunits. In the αβγδ nAChRs four rings composed of negatively charged and polar amino acid residues have been identified: the extracellular (20’), polar (2’), intermediate (-1’) and cytoplasmic (-5’) ring. These rings are depicted in fig. 1.9. Mutations in the intermediate ring appear to have more pronounced effect on single channel conductance and selectivity than mutations in any of the other rings, suggesting that the -1’residue might come into closer

Concatenation of the Cys-loop receptors ______contact with the permeating ions. This ring is believed to form a narrow region of the pore together with the polar ring in the cytoplasmic end of the M2 segment, which facilitates dehydration of cations and determines the ion flux and permeability of the channel (Imoto, 1993; Imoto et al., 1991).

As can be deduced from the alignment of M1-M2 and M2 amino acid sequences in fig. 1.10, the ring pattern of charged or polar residues is slightly different in the subunits of the anionic Cys-loop receptors. The negatively charged cytoplasmic ring at position -5’ is conserved, but there is no polar ring, and the intermediate and extracellular rings are composed of positively charged arginines found in the 0’ of all anionic subunits and in the 19’ position of all but the GlyR β subunits and the GABAA ρ1 subunits (Jensen et al., 2005). These charged residues located at each end of the M2 segment probably assist the permeation of anions. The ion selectivity-determining residues of anionic Cys-loop receptors therefore appear to be more widely spaced in the pore than is the case for their cationic counterparts.

It has been shown that point mutations involving the intermediate ring (-1’) in nAChR α7 subunits or 5- HT3A subunits can convert the channels from cation-selective to anion-selective. By inserting residues corresponding to those of anionic subunits in the -2’, -1’ and 13’ position of each subunit, it was demonstrated that the residues occupying these three positions were critical for the selectivity of the cationic channels

(Galzi et al., 1992; Gunthorpe and Lummis, 2001). Likewise, the homo-oligomeric GlyR α1 and GABAA ρ1 receptors could be switched from anionic to cationic by introducing the residues of cationic subunits into the - 2’, -1’ and 13’ positions, although loss of agonist sensitivity was observed for the mutant channels (Keramidas et al., 2000; Wotring et al., 2003). Thus, it was concluded that these three key amino acid positions located in the M2 segment and at the intracellular border of M2 were of crucial importance for the selectivity of both anionic and cationic homo-oligomeric Cys-loop receptors.

In an attempt to identify potential key residues for the ion selectivity of hetero-oligomeric Cys-loop receptors

Ahring and group replaced residues in the M1-M2 loop of GABAA α2, β3 and γ2 subunits with the corresponding residues of the nAChR α7 subunit (Jensen et al., 2002). In addition a set of “internal chimeras” were created in which the M2 segments of α2, β3 and γ2 were interchanged. Surprisingly, substitution of the

M2 segment of α2 or γ2 with that of α2, β3 or γ2 had no significant effect on the ion permeability of the chimaeric receptors, whereas substitution of the β3 segment with that of either α2 or γ2 yielded non-functional receptors. In addition, replacement of the M1-M2 loop sequence of the β3 subunit with that of α7 nAChR converted the ion selectivity of the chimaeric receptors from anionic to cationic. This was not the case for the

GABAA-nAChR α2 or γ2 chimeras. These results demonstrated an unequal contribution of the five subunits in the channel complex with respect to ion selectivity and proved that the position of the β3 subunit plays a unique role in determining the charge selectivity of hetero-oligomeric GABAA receptors (Jensen et al., 2005;

Concatenation of the Cys-loop receptors ______

Jensen et al., 2002). It remains to be established whether this asymmetrical position-dependent nature of the

GABAA α2β3γ2 ion selectivity filter is a common trait for the hetero-oligomeric Cys-loop receptors.

E K D Extracellular ring E K

T T G Polar ring T S Q E E Intermediate ring E E D K D Cytoplasmic ring D D

Fig. 1.9. The anionic rings contributing to the selectivity of cationic Cys-loop receptors. The pentameric arrangement of muscular nAChRs is represented in blue. The shape of the pore is outlined in red. The amino acid residues contributing to the anionic rings are denoted by single letters. K is positively charged; D and E are negatively charged; T, S and Q are polar, G is nonpolar. The figure is from Jensen et al.. (2003)

Cation conducting M1-M2 loop M2 channels

-5’ -1’ 2’ 13’ 20’ nAChR LPTDSG -EKMTLSISVLLSLTVFLLVIVELIPS nAChR LPPDAG2’ -EKMGLSIFALLTLTVFLLLLADKVPE 9’ nAChR LPADSG-EKTSVAISVLLAQSVFLLLISKRLPA nAChR LPAQAGGQKCTVSINVLLAQTVFLFLIAQKIPE nAChR LPAKAGGQKCTVAINVLLAQTVFLFLLAKKVPE nAChR 4 LPSECG-EKITLCISVLLSLTVFLLLITEIIPS nAChR 7 LPADSG-EKISLGITVLLSLTVFMLLVAEIMPA nAChR 2 LPSDCG-EKMTLCISVLLALTVFLLLISKIVPP

5-HT3AR LPPNSG-ERVSFKITLLLGYSVFLIIVSDTLPA 5-HT3BR LPPNCR-ARIVFKTSVLVGYTVFRVNMSNQVPR

Anion conducting channels

GlyR 1 INMDAAPARVGLGITTVLTMTTQSSGSRASLPK GlyR INPDASAARVPLGIFSVLSLASECTTLAAELPK

GABAAR 1-3,5 LNRESVPARTVFGVTTVLTMTTLSISARNSLPK GABAAR 2-3 INYDASAARVALGITTVLTMTTINTHLRETLPK GABAAR 1-2 INKDAVPARTSLGITTVLTMTTLSTIARKSLPK GABAAR 1 IDRRAVPARVPLGITTVLTMSTIITGVNASMPR

Fig. 1.10. Alignment of the M1-M2 loops and the M2 domains of selected Cys-loop subunits. All sequences are from human. The charged and polar rings are shown in blue. Both the cationic and the anionic receptors have a cytoplasmic ring (5’). The intermediate ring is located at -1’ for the cationic receptors and at 0’ for the anionic receptors, whereas the extracellular ring is located at 20’ for the cationic receptors and at 19’ for the anionic receptors. Only the cationic receptors have a polar ring (2’). The figure was modified from Jensen et al.. (2005).

Concatenation of the Cys-loop receptors ______

1.1.7 GABAA receptors

GABAARs are responsible for the majority of inhibitory neurotransmission in the mammalian brain (D'Hulst et al., 2009). Here, they mediate fast phasic inhibition at postsynaptic membranes and slower tonic inhibition at extrasynaptic and perisynaptic sites by transmembrane chloride conduction in response to the amino acid neurotransmitter GABA (Farrant and Nusser, 2005; Mody and Pearce, 2004). Due to their widespread localization GABAARs are involved in almost every physiological function of the brain, and serve as targets for several CNS drugs. The enhancement of GABA-mediated neuronal inhibition has proved to be a powerful therapeutic strategy for the treatment of neurological and psychiatric disorders such as anxiety, schizophrenia, epilepsy, insomnia and Huntington’s disease (D'Hulst et al., 2009).

GABAARs were initially identified pharmacologically as receptors that could be selectively activated by GABA and structural analogues such as muscimol, 4,5,6,7-tetrahydroisoxazolopyridin-3-o1 (THIP), piperidine-4-sulfonate (P4S), and isoguvacine. The receptors could be competitively inhibited by bicuculline, blocked by picrotoxin and modulated by benzodiazepines and barbiturates (Macdonald and Olsen, 1994).

GABAA receptors are also target for neurosteroids, anaesthetics and convulsants.

Today, 19 GABAA subunits have been cloned (table 1.1) and it has become increasingly clear that the mixed assembly of these 19 variants generates different receptor types that are confined to distinct cellular and subcellular locations in the brain and exhibit specific functions that can be selectively modulated by subtype specific drugs. Due to the differential compositions of these receptors, a compound can act as a full agonist at one receptor type and as a partial agonist or inverse agonist at other receptor types. For example muscimol is an agonist of the hetero-oligomeric GABAA receptors and a partial agonist of the homo-oligomeric GABAA receptors, whereas GABA acts as an agonist on the GABAA αβγ receptors and as a partial agonist on the

GABAA αβδ receptors (Belelli et al., 2009; Johnston, 2002).

A. Hetero-oligomeric GABAA receptors

As mentioned previously the most prominent GABAA receptor type in the mammalian central nervous system is composed of α, β and γ subunits in the stoichiometry 2:2:1. These, 75% contain α1-3/5 and γ2 subunits, and are sensitive to modulation by benzodiazepines (Olsen and Sieghart, 2008). Classical benzodiazepines such as diazepam, flunitrazepam, clonazepam and bromazepam are psychotropic drugs with anxiolytic and anticonvulsant effects. At higher concentrations they display a sedative, hypnotic and muscle relaxant effect (Sieghart and Sperk, 2002). They bind allosterically to a site distinct from the agonist binding site on the extracellular domain of αβγ receptors and enhance neuronal inhibition by increasing the frequency of channel openings mediated by the agonist GABA (Sigel, 2002). The benzodiazepine binding pocket is located at the interface of the α1-3/5(+) and (-)γ2 subunits analogous to the GABA binding pocket at the interface of β(+) and

Concatenation of the Cys-loop receptors ______

(-)α subunits, and therefore only the γ2 containing GABAA receptors are benzodiazepine-sensitive.

Furthermore the α4 and α6 containing GABAA receptors show very little sensitivity towards the classical benzodiazepines (Sigel, 2002). The benzodiazepine binding site can also bind negative allosteric modulators such as methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) which reduce the GABA- evoked currents, or null modulators such as flumazenil which act as antagonists of the benzodiazepine site (D'Hulst et al., 2009). The relative positions of the GABA binding sites and the benzodiazepine binding site are shown in fig. 1.11A.

Benzodiazepine-insensitive α1βδ, α4βδ and α6βδ receptors have been localized in hippocampal interneurons, forebrain and cerebellar granule cells, respectively (Olsen and Sieghart, 2008). These δ-containing receptors appear to be responsible for tonic GABA-evoked inhibition extrasynaptically, and show high sensitivity towards GABA and neurosteroids such as pregnanolone and (3α,5α)-3,21-dihydroxypregnan-20-one

(3α5αTHDOC) (Olsen and Sieghart, 2008). Steroids also modulate other GABAA receptor types, and they can be both enhancers and depressors of GABA-evoked currents (Akk et al., 2007). Additionally, at high concentrations potentiating steroids are able to function as agonists themselves and activate receptors in the absence of GABA. Examples of positive steroid modulators are alphaxolone and ganaxolone (D'Hulst et al., 2009).

Barbiturates such as pentobarbital, phenobarbital and secobarbital have been shown to allosterically enhance GABA-evoked currents by increasing the duration of channel openings. At concentrations above 50 mM they are able to activate GABAA receptors in the absence of GABA as is the case for high concentrations of neurosteroids (Serafini et al., 2000). The sensitivity towards barbiturates appears to reside within the β subunit of GABAA receptors since splicing of the M3 transmembrane segment of β3 subunits into the barbiturate-insensitive GABAA ρ1 subunit produced receptors that could be modulated by pentobarbital (Johnston, 2002).

In summary, hetero-oligomeric GABAA receptors serve as a common target of several general anaesthetics, and obtaining knowledge about their structure and function is crucial for the improved treatment of various CNS-related diseases.

B. Homo-oligomeric GABAA receptors

In 1991 Cutting et al. applied PCR to isolate a cDNA encoding an unknown GABAA subunit from a human retinal cDNA library. The cDNA gave rise to a polypeptide of approximately 458 amino acid residues which displayed 30-38% similarity to the known GABAA α, β, γ and δ subunits. The subunit was named GABAA ρ1

(Cutting et al., 1991). Upon expression in Xenopus oocytes, ρ1 produced GABA induced chloride currents with dose-response relationships mimicking those of previously cloned bovine GABAA α and β subunits.

Concatenation of the Cys-loop receptors ______

However, in contrast to the other GABAA subunits, ρ1 was able to assemble into homo-oligomeric channels, and did not seem to coassemble with GABAA α or β subunits (Hackam et al., 1998; Koulen et al., 1998;

Shimada et al., 1992). ρ1 channels possessed higher sensitivity to GABA and lower sensitivity to muscimol than most of the known GABAA receptors, and their expression seemed predominantly restricted to the retina

(Cutting et al., 1991; Shimada et al., 1992). A schematic representation of the homo-oligomeric GABAA ρ1 receptor is shown in fig. 1.11B.

Today, three GABAA ρ subunits have been identified (GABAA ρ1-3) by cloning of mammalian retinal cDNA libraries (Lopez-Chavez et al., 2005; Ogurusu and Shingai, 1996; Wang et al., 1994). These subunits assemble into homo-oligomeric or pseudo hetero-oligomeric receptor complexes (the latter composed of different ρ isoforms) which display functional and pharmacological traits corresponding to the phenotype of a non-conventional GABA receptor type called GABAC (Enz and Cutting, 1999; Shimada et al., 1992). GABAC was initially defined as a bicuculline- and baclofen-insensitive ionotropic GABA receptor found in vertebrate neurons of cat spinal cord and various invertebrate tissues as well as in oocytes injected with bovine retinal mRNA and in the retinae of white perch and rat (Feigenspan and Bormann, 1994; Feigenspan et al., 1993;

Woodward et al., 1992, 1993). GABAC currents were characterized by slow activation and deactivation kinetics, by a low degree of desensitization, and by insensitivity to modulation by benzodiazepines and barbiturates (Amin and Weiss, 1994; Chang and Weiss, 1999). The receptors could be selectively activated by the cis-enantiomer, CACA, of the GABA analog 4-aminocrotonic acid, and were competitively antagonized by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA) and by THIP (Johnston, 2002).

Electrophysiological species-specific comparison of native retinal GABAC receptors and heterologously expressed cloned retinal ρ subunits confirm that GABAC receptors are likely to be composed of ρ subunits (Qian et al., 1997; Zhang et al., 1995). This has also been supported by in situ hybridization and antibody labeling studies that localized the expression of ρ1 mRNA to bipolar cells which displayed GABAC-like pharmacological responses (Enz and Bormann, 1995; Enz et al., 1996; Yeh et al., 1996), and by a knock-out experiment which demonstrated elimination of GABAC responses in rod bipolar cells upon inactivation of the

ρ1 gene in rats (McCall et al., 2002). The correlation between GABAC receptors and ρ subunits has started an ongoing debate on the classification of this receptor type. On one side it is argued that the distinct pharmacological profile should place it in a class of its own, GABAC (Boorman et al., 2000). On the other side, multiple sequence alignment and phylogenetic analysis categorize ρ subunits as belonging to the

GABAA family, although more distantly related than other GABAA subunits. The International Union of

Pharmacology committee proposes the term GABAAOr to specify the insensitivity to benzodiazepines (0) and the ρ subunit composition (r) (Barnard et al., 1998). Here, the ρ containing receptors will simply be referred to as GABAA ρ receptors.

Concatenation of the Cys-loop receptors ______

Since their discovery, GABAA ρ receptors have been detected in many areas of the central nervous system such as the superior colliculus, thalamus, hippocampus and pituitary (Enz and Cutting, 1999; Koulen et al.,

1997; Mejia et al., 2008). In the retina, the GABAA ρ1 receptors are highly expressed on the axon terminals of bipolar cells where they seem involved in the regulation of visual signaling by modulating the release of excitatory transmitter to amacrine- and ganglion cells (Lukasiewicz et al., 1994; Lukasiewicz and Werblin,

1994). Besides, GABAA ρ1 receptors have been implicated in sleep-wake regulation (Arnaud et al., 2001), memory and learning mechanisms, pain perception and regulation of hormone secretion (Chebib et al., 2009; Gibbs and Johnston, 2005).

B Fig.B 1.11. Schematic representation of A B A: the GABAA αβγ receptor and B: the GABAA ρ receptor. For simplicity only the pore-lining M2-segment is shown in

+ dark red. The subunit interfaces participating in binding of GABA are GABAA GABAGABAA GABA A marked with red. TheA blue arrows denote

αβγ α/β ρ α/β

2 2

β GABA binding sites, whereas the red

2 2

β β

2 2 arrow denotes the benzodiazepine binding site. The ρ receptor is insensitive to benzodiazepines and can be activated with less than five intact GABA binding sites as described in section 1.1.6B.

C C D D γ α_ γ α_ Table 1.2 summarizes some of the general propertiesγ α_ of homo-oligomeric and heteroγ -oligoα_ meric GABA + + A β β β β + + + β+ β β β receptors. - + - + α -αα - α β-α + γ βγ--αβ-+α γ+ β-α γ-β-α + β-α

E EF γ α_ F γ α_ α β α_ α β α_ + + + β β + β β β β +β β + + +- - - α α- α α γ γ

β-α βγ--αβ-α γ-β-α

G G +β +β _ _ α β α α β α α _ α _ _ + _ + β+ α β β β+ α β β + + - α - α

β-α β-α

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Concatenation of the Cys-loop receptors ______

Fuctional parameter Homo-oligomeric GABAARs Hetero-oligomeric GABAARs

GABA EC50 ~1-5 μM 5-100 μM Hill slope 1.6 – 3.3 2 Activation/inactivation Slow Fast Desensitization Weak Strong Conductance 7 pS 27-30 pS Open time 150-200 ms 25-30 ms Selectivity Anions (Cl-) Anions (Cl-) Pore size 5.1 Å 5.6 Å

Pharmacology Agonists/ GABA GABA CACA TACA Partial agonists TAMP TAMP CAMP Muscimol Muscimol Isoguvacine Isoguvacine THIP I4AA P4S 4-PIOL

Antagonists I4AA Bicuculline TPMPA Picrotoxin THIP SR-95531 Picrotoxina Gabazine TACAc TBPS

Modulators Neurosteroids Benzodiazepinesb Triazolopyridazinesb Imidazopyridinesb Barbiturates Neurosteroids

a Table 1.2. General physiological and pharmacological properties of GABAA receptors. Strong b C antagonist for only the ρ1 receptors. Do not modulate receptors containing α4/6, δ or ε subunits. ρ1 antagonist, ρ2 partial agonist. Abbreviations: GABA, γ-aminobutyric acid; CACA, Cis-4-amino-crotonic acid; CAMP, Cis-2-aminomethyl-cyclopropane carboxylic acid; I4AA, Imidazole-4-acetic acid; P4S, Piperidine -4-sulfonic acid; 4-PIOL, 5-(4-piperidyl)isoxazol-3-ol; TACA, Trans-4-aminocrotonic acid; TAMP, Trans-2-aminomethyl-cyclopropane carboxylic acid; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4- c]pyridine -3-ol; TPMPA, (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid. (Bormann, 2000; Frolund et al., 2002; Johnston, 2002).

Concatenation of the Cys-loop receptors ______

1.2 CONCATENATION

As outlined above much knowledge about the Cys-loop receptors has been extracted by heterologous expression of different subunit combinations in mammalian cells or Xenopus oocytes. This strategy is however limited by the fact that the expression of a mixture of single subunits involves the potential risk of mixed assembly resulting in the generation of more than one receptor subtype. The concatenation technique aims to overcome this problem by providing a means of effectively constraining the subunit stoichiometry and arrangement of recombinant receptors. The technique can be applied as a molecular tool to study multimeric transmembrane proteins that contain the N- and C-terminal on the same side of the membrane. By use of subcloning procedures, the subunit cDNAs are covalently linked in sequence while maintaining a single open reading frame to create one large fusion protein in which the N- and C-terminal of tandem subunits are connected by a linker sequence. This enables the expression of receptors with a predefined stoichiometry and arrangement. Detailed description of the experimental strategy underlying this technique is given in the Experimental procedures section. Composition of the linker sequence will be described in section 1.3.3.

1 2 3 4 5

M1M21M3M4 M1M22M3M4 M1M23M3M4 M1M24M3M4 M1M25M3M4

Fig. 1.12. The principle of concatenation. Top: 1-5 symbolize the cDNAs of 5 five subunits from N to C. The coding regions have been fused by a linker 4 sequence which is in dotted red. Middle: schematic drawing of the fusion 1 protein, a penta-concatamer. The five subunits are connected by the linker from their C- to N- terminal protrusions. The dark red region symbolizesρ1 the pore- 3 lining M2 segment. Bottom: Top view of linked pentameric arrangement. The 2 figure was modified from Jensen et al. (2003). ρ 1

1.2.1 Applications The concatenation technique was introduced in 1990 by Isacoff et al. who sought to determine the 10 composition of Shaker A-type K+ channels expressed in Xenopus oocytes (Isacoff et al., 1990). Cloning of the Drosophila Shaker gene in the 1980s had revealed a structural resemblance between the α subunit of the Shaker K+ channel and each of the four homologous domains I-IV of the Na+ channel α subunit. This

Concatenation of the Cys-loop receptors ______prompted to the speculation whether K+ channels were actually composed of four homologous subunits. Isacoff and collegues tested this hypothesis by linking two variants of the Shaker gene, ShA and ShB. The C- terminal of ShA was fused with the N-terminal of ShB by a stretch of 10 glutamine residues and the resulting tandem construct was functionally characterized in Xenopus oocytes by patch clamp measurements. Currents kinetics and single-channel conductances of the tandem were compared to those of co-injected ShA/ShB, and to those of separately injected ShA and ShB. Results revealed that currents measured from co-injected or tandem-injected oocytes were different from pure ShA or ShB currents, and did not correspond to the sum of these two variants. This led to the conclusion that ShA and ShB probably co-assembled into a multimeric K+ channel composed of an even number of similar or identical subunits (Isacoff et al., 1990).

Since then, the concatenation technique has been applied to study the voltage-gated and inwardly-rectifying K+ channels (Hurst et al., 1992; Lee et al., 1994; Liman et al., 1992; McCormack et al., 1992; Pessia et al., 1996; Tytgat et al., 1993) and a wide variety of other proteins such as the E. coli lactose permease (Sahin- Toth et al., 1994), the cyclic nucleotide-gated ion channels (Shapiro and Zagotta, 1998; Varnum and Zagotta, + 1996), the epithelial sodium channel (Firsov et al., 1998), the renal type IIa Na /Pi cotranorter (Kohler et al.,

2000), the cystic fibrosis transmembrane conductance regulator (Zerhusen et al., 1999), the P2X receptors (Newbolt et al., 1998; Nicke et al., 2003) and the Cys-loop receptors as described in details in the following section.

1.2.2 Concatenation studies of Cys-loop receptors

A. Stoichiometry

In 2001 Sigel and group synthesized concatenated dimers of GABAA α1 and β2 subunits to determine the stoichiometry of GABAA α1β2 and α1β2γ2 receptors. When dimers were expressed alone or in combination, almost no current was observed, as was the case for dimers expressed with single α1 subunits. In contrast, co- expression of dimers with single β2 or γ2 subunits yielded functional receptors with maximal current amplitudes and apparent GABA affinities similar to those of the wildtype receptor (Baumann et al., 2001).

Based on these results it was concluded that receptors produced from GABAA α1 and β2 subunits were not composed of an even number of subunits, an observation supported by earlier immunoprecipitation studies that indicated a pentameric arrangement of the receptor (Im et al., 1995; Tretter et al., 1997). Furthermore receptors were proposed to contain two α1 subunits and three β2 subunits for the α1β2 receptors, and two α1 subunits, two β2 subunits and one γ2 subunit for the α1β2γ2 receptors (Baumann et al., 2001).

In 2002 the Sigel group combined the dual constructs with triple constructs of GABAA α1, β2 and γ2 subunits linked in different orders. The dimers and trimers were expressed pairwise in Xenopus oocytes and were subjected to functional characterization by patch clamp measurements. Only subunit combinations resulting in

Concatenation of the Cys-loop receptors ______the pentameric arrangement γ2β2α1β2α1 gave rise to currents of wildtype magnitude and showed wildtype-like sensitivity towards GABA, bicuculline and diazepam. This led the group to suggest a subunit order of

γ2β2α1β2α1 around the channel pore of GABAA α1β2 γ2 receptors (Baumann et al., 2002).

Lindstrom and group concatenated dimers of nAChR α4 and β2 subunits and co-expressed these with either free α4, β2 or β4 subunits to biophysically characterize the resulting receptors. They observed that the (α4)3(β2)2 stoichiometry produced more agonist-evoked current per surface expressed receptor than did the (α4)2(β2)3 stoichiometry whereas the (α4)3(β2)2 stoichiometry produced receptors which were less sensitive to acetylcholine. This confirmed the hypothesis of two nAChR α4β2 populations of distinct stoichiometry which are distinguishable by low and high agonist affinity as described in section 1.1.4A (Zhou et al., 2003). Six years later, Bermudez and group concatenated α4β2 nAChRs into the pentameric arrangements: β2-α4-β2-α4-β2 and β2-α4-β2-α4-α4 to demonstrate stoichiometry-specific potencies of the α4β2 nAChR agonists A85380, 51- A85380, cytisine, epibatidine, TC2559 and the competitive antagonist dihydro-β-erythroidine (Carbone et al., 2009).

The stoichiometry of hetero-oligomeric GlyRs has also been investigated by concatenation. These receptors have been proposed to contain two α subunits and three β subunits by some groups, whereas others have found indications of a stoichiometry of three α subunits and two β subunits (section 1.1.4D). To solve this discrepancy Grudzinska et al. (2005) linked the C-terminus of an α subunit to the N-terminus of a β subunit and co-expressed this construct with either free mutant α or wildtype β subunits. Co-expression with the α subunit resulted in receptors with pharmacological properties corresponding to those of a homo-oligomeric GlyR of exclusively α subunits whereas co-expression with the β subunit yielded receptors with similar agonist sensitivity as the wildtype αβ GlyRs. This led them to suggest that the stoichiometry of hetero- oligomeric GlyRs was (α)2(β)3 (Grudzinska et al., 2005).

Overall, these studies demonstrate the unique insight into Cys-loop receptor stoichiometry and arrangement that can be obtained with successful concatenation.

B. The functional role of subunits Concatenation has also been applied to elucidate the role of distinct subunits or subunit isoforms in Cys-loop receptors. In 2004 Minier and Sigel used concatenated dimers and trimers to show that α6 containing αxβ2γ2

GABAA receptors were more sensitive to GABA than the α1 containing receptors, and that a single α6 subunit in the pentamer was enough to confer sensitivity to the α6-specific noncompetitive inhibitor, furosemide.

Furthermore, they demonstrated that the presence of one α1 subunit (but not an α6) was enough to enable allosteric potentiation by diazepam, as long as the neighbouring subunit was γ2 (Minier and Sigel, 2004a;

Sigel et al., 2006). Analogously another concatenation study by the Sigel group investigated the role of β1 and

Concatenation of the Cys-loop receptors ______

β2 subunits present in the same GABAA α1βxγ2 receptor complex and showed positional insensitivity of stimulation by the β2-selective agents loreclezole and etomidate (Boulineau et al., 2005). Recently, the incorporation and functional profile of δ subunits in GABAA receptors was examined. Dimers, trimers and a pentamer were designed from GABAA α6β3δ subunits to determine the position of the δ subunit in the receptor and measure the sensitivity towards neurosteroids and ethanol. The results indicated that the δ-containing receptors were not sensitive to ethanol, and in contrast to γ subunits, δ could occupy multiple positions in a

GABAA α6β3δ receptor. Furthermore a subunit combination, α1-β3-α1/β3-δ (trimer + dimer), which showed little response to GABA was uncovered by co-application with the neurosteroid THDOC, indicating that some extrasynaptic receptors may not exert their inhibitory effect on neuronal activity unless stimulated by both GABA and neurosteroids (Baur et al., 2009; Kaur et al., 2009). This exemplifies the usefulness of the concatenation technique in studies of subunits that are difficult to distinguish or characterize functionally in untethered, heterologously expressed receptors.

C. Positional effects of mutations

In 2004 Gallagher et al. created dual and triple constructs of GABAA α1β2γ2 receptors to study the positional effect of a missense mutation A322D found in the α subunit gene GABRA1 of patients suffering from an autosomal dominant form of Juvenile myoclonic epilepsy (JME). The patients were heterozygous for this mutation and therefore four different GABAA α1β2γ2 receptors could be generated from their wildtype and mutant alleles: a wildtype receptor, a receptor containing one mutation in either of the two α subunits, or a homozygous receptor. The group used concatenated construct to show that heterozygous receptors in which the mutated α subunit was situated between β and γ (Hetβαγ) had lower peak current amplitudes and reduced expression in HEK293T cells compared to receptors in which the mutated subunit was situated between two β subunits (Hetβαβ), indicating an asymmetrical effect of the mutation at the two α subunits (Gallagher et al., 2004).

In another positional study, Sigel and group introduced mutations in the α and β subunits of concatenated

GABAA α1β2γ2 concatenated dimers and trimers to elucidate the pharmacological properties of the two agonist binding sites of a receptor. The results indicated that both sites must be occupied by agonist for efficient gating of the receptor, and that bicuculline occupancy of a single site is sufficient to inhibit channel opening. Furthermore it was shown that mutation of the binding site flanked by γ and α in a counterclockwise γ-β-α-β- α arrangement (site 2) had a more dramatic effect on the GABA sensitivity of the mutant receptors than mutation of the binding site flanked by γ and β in a γ-β-α-β-α arrangement (site 1). Finally the agonist muscimol displayed a higher apparent affinity for site 1 (Baumann et al., 2003). The same mutational strategy was used by Baur and Sigel in concatenated dimers and trimers of GABAA α1β2γ2 subunits to selectively disrupt the agonist affinity of each binding site. The mutated receptors were characterized with respect to

Concatenation of the Cys-loop receptors ______diazepam sensitivity to show that the presence of one functional agonist binding site was sufficient for potentiation by benzodiazepines (Baur and Sigel, 2005).

In 2006 Sivilotti and group concatenated all five subunits of a nAChR α3β4 receptor and introduced the reporter mutation L9’T in the M2 segment of either an α3 or a β4 subunit, which has been shown to increase the agonist sensitivity of the receptor. Concentration-response curves with acetylcholine showed that the mutation had a greater effect when carried by the α3 subunit than the β4 subunit, and indicated that the pore- lining domains of the two α3 and the three β4 subunits provided nonequivalent contribution to receptor gating (Boorman et al., 2000; Groot-Kormelink et al., 2006).

In summary, it is evident that the concatenation technique has been a valuable tool in several Cys-loop receptor studies to elucidate functional and pharmacological properties of different positions within the pentameric receptors.

1.2.3 Linker design A variety of different sequences have been used to link the two cDNAs of a tandem construct, as shown in table 1.3. In each case, the purpose of such a linker stretch was to position the tandem subunits next to each other in the resulting receptor for the predetermination of subunit arrangement and stoichiometry. Many concatenation studies were performed with linkers of 8-10 glutamines (grey section of table 1.3). It seems that at this length, polyglutamine is hydrophilic and adopts a random coil conformation that confers flexibility and extensibility to the linker (Altschuler et al., 1997). However, it has been speculated that translation of a long polyglutamine linker might exhaust the pool of relevant tRNA and result in premature termination of the synthesized protein. Furthermore, the Sigel group questioned the presence of a signal peptide in the N- terminal of the second subunit in a tandem, and argued that this hydrophobic sequence of 20-30 amino acids in the middle of the synthesized fusion protein could have unpredictable effects on concatamer assembly and expression (Baumann et al., 2001).

To elucidate the importance of proper linker design, two linker optimization studies were conducted. In 2001 the Sigel group set out to determine the minimal linker length for the synthesis of functional GABAA α1β2 concatamers. Two tandem constructs were made: α1-β2 and β2-α1 with 3 and 4 different linker lengths, respectively. The constructs were linked with different lengths due to differences in the predicted number of C-terminal amino acids after the fourth transmembrane domain, and differences in the number of N-terminal amino acids preceeding the first transmembrane domain. α1-β2 was linked with 0, 7 or 10 glutamine residues, and β2- α1 was linked with 10, 15, 20 or 23 glutamine, alanine and proline residues. For all constructs, the N- terminal precursor form was used in the first tandem subunit to ensure signal sequence mediated translocation of the protein, while the second subunit was depleted of its signal sequence. The tandem constructs were co-

Concatenation of the Cys-loop receptors ______expressed with free β2 or γ2 subunits in Xenopus oocytes and the maximal current amplitude and apparent GABA affinity was measured and compared to that of the wildtype. The minimal linker lengths that generated functional concatamer receptors were found to be 10 amino acid residues for the α1-β2 construct and 23 amino acid residues for the β2-α1 construct, although α1-7-β2 and β2-20-α1 were almost as functional. Shorter linkers gave rise to altered GABA affinity and lower maximal current amplitudes (Baumann et al., 2001). The same linker optimization strategy was applied to determine the linker lengths required to express γ-β, α-γ and γ-α for the synthesis of trimeric constructs; γ-α-β, γ-β-α and β-α-γ (Baumann et al., 2002) and for the synthesis of dimers and trimers consisting of α1, β3 and δ subunits (Kaur et al., 2009). The linkers used by the Sigel group are shown in blue in table 1.3.

Lindstrom and group also performed a linker optimization experiment on dimeric nAChR α4β2 constructs linked with either 0, 6 or 12 repeats of alanine, glycine and serine residues. Sucrose gradient sedimentation of concatenated constructs revealed that dimers with a total linker length of 50 amino acid residues from the end of the M4 transmembrane domain of the first subunit to the N-terminus of the second subunit (signal peptide removed) were too long to constrain the orientation and assembly of subunits resulting in receptors with more than five subunits. On the other hand linkers of 25 amino acid residues seemed to inhibit the assembly and surface expression of concatenated constructs alone but allowed function upon co-expression with free subunits (Zhou et al., 2003). This demonstrates the importance of linker design for proper expression of the concatenated constructs.

Study Expression Receptor Amino acid sequence of linker Conclusion system

Baur et al. XO GABAA α6 β3δ α6-11-β3: Q5TGQ4, Same conclusions as in Baur et al., 2009, but for GABA Rs containing 2009 (dimers + trimers) α6-11-δ: Q5TGQ4 A the α6 subunit instead of α1. β3-23-δ: as in Bolineau et al., 2005 β3-26-α6, δ-26-α6, δ-26-β3 as in Baumann et al. (2002) Kaur et al. XO GABAA α1 β3δ α1-10-β3, α1-10-δ, β3-23-α1, δ-23-α1 as The δ subunit can occupy multiple 2009 (dimers, trimers, in Bolienau et al. (2005) positions in GABAA α1β3δ receptors and may participate in the formation pentamers) β3-26-δ: as in Baumann et al. (2002) of an agonist binding site. Ethanol does not modulate these receptors. Baur et al. XO GABAA α1β2γ2 α1-10-β2, α1-10-γ2, β2-23-α1 as in The five subunits of the GABAA 2006 (pentamer) Baumann et al. (2001) α1β2γ2 receptor can be concatenated to yield a functional receptor. γ2-26-β2: Q5A3PAQ3APA3PA2Q5 Bolineau et XO GABAA α1β1/2γ2 α1-10-β1: Q4TGQ4 (e) GABAARs containing β1 and β2 subunits in the same pentamer are al. 2005 (dimers + trimers) β1-23-α1: Q5A3PTGQA3PA2Q5 directly gated by etomidate similar to γ2-26-β1: as in Baumann et al. (2002) receptors containing only the β1 isoform and the effect of etomidate is independent on the relative positions of the two β subunit isoforms in a pentamer. Minier and XO GABAA α1/6β2γ2 β1-23-α1, β1-23-α6, γ2-26-β1 A single α6 subunit in α1/6β2γ2 Sigel 2004 (dimers + trimers) As in Baumann et al. (2001, 2002) GABAARs is sufficient to confer high furosemide sensitivity and the diazepam efficacy is determined exclusively by the α subunit neighbouring the γ2 subunit.

Concatenation of the Cys-loop receptors ______

Gallagher et HEK293T GABAA α1β2γ2 β1-23-α1, γ2-26-β1 α1β2γ2 GABAAR currents in al. 2004 (dimers + trimers) As in Baumann et al. (2001, 2002) individuals suffering from juvenile myoclonic epilepsy likely result primarily from wild-type and heterozygous GABAARs with the α1(A322D) mutation located between two β subunits. Baumann et XO GABAA α1β2γ2 α1-10-β2, β2-23-α1, γ2-26-β1 The binding of two agonist molecules al. 2003 (dimers + trimers) As in Baumann et al. (2001, 2002) to an α1β2γ2 GABAAR is required for proper activation, and GABA, muscimol and bicuculline show differential preference for each of the two binding sites. Baumann et XO GABAA α1β2γ2 α1-10-β2, α1-10-γ2, β2-23-α1, γ2-23-α1 as α1β2γ2 GABAARs are arranged in the al. 2002 (dimers + trimers) in Baumann et al. (2001) counterclockwise order γ-β-α-β-α when viewed from the synaptic cleft. γ2-26-β1: Q5A3PTGQ2AQA2PA2Q5 Baumann et XO GABAA α1β2 and α1-0-β2: 0 A pentameric arrangement containing two α and three β subunits or two α , al. 2001 α1β2 γ2 α1-7-β2: Q7 1 2 1 two β2 and one γ2 is proposed for the (dimers) α1-10-β2: Q10 β -10-α : Q GABAA receptors composed of α/β 2 1 10 and α/β/γ subunits, respectively. β2-15-α1: Q5A3PAQ5 β2-20-α1: Q5(A3P)2A2Q5 β2-23-α1: Q3(Q2A3PA)2AQ5 Carbone et XO nAChR α4β2 β2-6-α4: (AGS)6 The β-α-β-α-β and β-α-β-α-α penta- concatamers are valid models of al. 2009 (pentamers) α4-9-β2: (AGS)9 (α4)2(β2)3 and (α4)3(β2)2 receptors, respectively. nAChR α4β2 preferring compounds display stoichiometry- specific potencies. Zhou et al. XO nAChR α4β2 α-12-β: QEGT(AGS)12TG The nAChR (α4)3(β2)2 stoichiometry produces greater agonist-induced 2003 (dimers) β-6-α: EG(AGS)6 currents per surface nAChR than the α-6-β: QEGT(AGS)6TG β-α: EG (α4)2(β2)3 stoichiometry, and displays lower sensitivity towards the agonist.

Bolieau et HEK293 GABAA α1β2γ2 α-9-β: Q9 GABARAP does not alter receptor al. 2005 (dimer) kinetics directly but facilitate the surface expression of α1β2γ2 GABAARs. Groot- XO nAChR α3β4 β-8-α:Q8 The five subunits of the α3β4 nAChR can be concatenated to yield a Kormelink (pentamer) α-8-β:Q8 functional receptor. et al. 2006 Groot- XO + nAChR α2/4/6 β2/4 β-8-α:Q8 nAChRs composed of β4-α3 tandems co-expressed with Kormelink HEK293 (dimers) α-8-β:Q8 et al. 2004 free β4 subunit are heterogeneous. A proportion of these channels incorporate two or three β monomers. Im et al. HEK293 + GABAA α6β2γ2 α-10-β:Q10 GABAARs are pentamers consisting 1995 Sf-9 (dimers) of two α6, two β2, and one γ2 for the α6β2γ2 and three α6 and two β2 for the α6β2 subtype. Hurst et al. XO Kv1.1 (RBK1) RBK1-10-RBK1: Q10 Voltage-dependent potassium 1992 (tetramer) channels are tetrameric and the four subunits interact cooperatively during channel activation. + Isacoff et XO K Shaker ShB-10-ShB: Q10 The Shaker A-type potassium channels are heteromultimeric. al. 1990 (dimers) ShB-10-ShA: Q10 ShA-10-AhB:Q10 Nicke et al. XO P2X1R P2X1-7- P2X1: Q5 The P2X receptors are probably 2002 (dimer, trimer, trimeric. Formation of lower order tetramer, by-products can be a pitfall of the pentamer, concatamer approach.

hexamer) Table 1.3. Overview of linkers used in a subset of previous concatenation studies. The number between subunits such as β-23-α denotes the number of residues linking the two subunits from C’ to N’. XO: Xenopus Oocytes, HEK293: Human Embryonic Kidney Cell line 293. Sf-9: Spodoptera frugiperda insect cell line 9. The amino acid sequences of linkers are given by the single letter code. The blue shading shows studies with linker sequences based on Bauman et al.. (2001, 2002). The grey shading shows studies with linker sequences of 5-10 glutamines. The blue and white rows represent studies in which the signal peptide was removed from the second subunit in a tandem. 29

Concatenation of the Cys-loop receptors ______

1.2.4 Precautions in the use of concatenation Although concatenation has been applied successfully in many studies to express receptors of defined architecture the reliability of this technique has been questioned in a number of cases. For example Lindstrom and group observed that dimeric α4β2 constructs with sufficiently long linkers could assemble into functional dipentamers or into monopentamers with a dangling subunit protruding from the pentameric complex (Zhou et al., 2003). Sivilotti and group observed that some β4-α3 dimers were able to assemble into functional channels when expressed in oocytes alone, although the current level was much lower than that of dimers co- expressed with free β4 subunits (Groot-Kormelink et al., 2004). This phenomenon was also seen with GABAA dimer β3-α1 and trimer β3-α1-δ in a study on δ-containing GABAA receptors (Kaur et al., 2009), and with trimers of the tetrameric GIRK channels (Silverman et al., 1996). Furthermore, results obtained by the use of reporter mutations have implied that some tandem constructs are incompletely incorporated into the functional receptors (Groot-Kormelink et al., 2004; McCormack et al., 1992). The expression of monomeric and dimeric byproducts generated from higher-order concatamers has been reported for the P2X1 receptor, as well as the intracellular aggregation of concatameric constructs in E.R (Nicke et al., 2003). These findings indicate that concatenation studies should be interpreted with caution, unless combined with biochemical experiments for validation. A number of approaches have been applied to check for artefacts such as linker proteolysis, incomplete subunit incorporation and unpredicted rearrangements of tandems. These include:

Western blot: to check for efficient translation or potential degradation of concatenated constructs (Baumann et al., 2001, 2003; Boileau et al., 2005; Gallagher et al., 2004; Groot-Kormelink et al., 2004; Newbolt et al., 1998; Zhou et al., 2003) Sucrose gradient sedimentation and blue native PAGE analysis: to demonstrate the size of assembled concatamers in the membrane or intracellularly (Nicke et al., 2003; Zhou et al., 2003) Pharmacological assays: to test whether expressed concatameric constructs bind and respond to agonists, competitive antagonists and modulators in a manner similar to the wildtype receptor. This also indicates whether rearrangements or interspersing of tandem subunits occur (Baur et al., 2006; Baur and Sigel, 2005; Boulineau et al., 2005; Kaur et al., 2009; Minier and Sigel, 2004a) Radioimmunoassays: to determine surface- or intracellular expression of the concatenated constructs, and assess the ability of concatenated receptors to form binding sites (Im et al., 1995; Zhou et al., 2003) Glycosylation assays: to test for proper transmembrane folding or E.R. retention of concatenated constructs (Nicke et al., 2003) Incorporation of reporter mutations: to investigate whether tandem constructs are properly incorporated into the receptors (Groot-Kormelink et al., 2006; Groot-Kormelink et al., 2004; McCormack et al., 1992).

Concatenation of the Cys-loop receptors ______

In the majority of reported cases, though, the concatenation technique has proven successful and revealed many details of receptor architecture and function, which are difficult or impossible to identify by conventional heterologous co-expression of free subunits. The technique has contributed in large scale to the current knowledge about the properties of different Cys-loop subunit classes and isoforms, and mapped the stoichiometry and arrangement of various hetero-oligomeric receptor types. However, no concatenation studies of homo-oligomeric Cys-loop receptors have been published to date. This is intriguing since a homo- oligomeric receptor of five tethered subunits could provide new insights into conserved properties of the Cys- loop subunits much like the structurally related homo-oligomeric AChBP.

______

2. AIM

The initial goal of this project was to create a homo-oligomeric GABAA ρ1 penta-concatamer which could be site-specifically modified to elucidate positional effects of mutations. Ideally, this construct could serve as a model system for the Cys-loop receptors to study potential asymmetries in the contribution of identical subunits to mechanisms such as agonist binding and selectivity filtering of ions. More specifically, the agonist binding sites of a penta-concatamer of identical subunits could be selectively mutated to establish the number of bound agonist molecules required to fully activate a homo-oligomeric receptor, and to demonstrate whether some of the five binding sites play greater roles with respect to activation. Furthermore, a homo-oligomeric penta-concatamer could potentially serve to clarify whether the charge selectivity of Cys-loop receptors is asymmetrically allocated to different positions in the pentameric complex relative to the agonist binding sites, as indicated by Jensen et al. (2002) and described in section 1.1.6D. The strategy of the project was:

To identify an optimal linker for the concatenation of GABAA ρ1 subunits.

To tether the five subunits of a GABAA ρ1 receptor with this optimal linker.

To assay the functional expression of the GABAA ρ1 penta-concatamer in CHO cells. If the previous aims were successfully fulfilled, to identify the number of agonist molecules required

to fully activate the penta-concatenated GABAA ρ1 receptor.

A second aim of the project was to functionally characterize previously synthesized hetero-oligomeric

GABAA concatamers in Xenopus oocytes. These concatamers are currently applied for studies of GABAA receptors at NeuroSearch and similar constructs have been used by the Sigel group in an array of studies as shown in table 1.3 (blue shading). However, our lab has observed some unexpected behaviours of these constructs which remain to be documented and clarified, namely the generation of GABA-evoked current by the separate expression of dimer and trimer concatamers. Therefore, the strategy of this part was:

Concatenation of the Cys-loop receptors ______

To determine the functional expression and sensitivity towards GABA and diazepam of the hetero-

oligomeric GABAA β3-α1 dimers and γ2-β3-α1 trimers expressed separately, together or in combination with free γ subunit in Xenopus oocytes.

To evaluate whether the hetero-oligomeric GABAA concatamers provide a reliable means of expressing receptors of defined stoichiometry and arrangement.

Both studies were based on previous work in our lab. Five GABAA ρ1 subunits of identical amino acid sequence, distinguishable only by the presence of two unique restriction sites in each subunit, were constructed in a master thesis project by Ditte Dibber in 2008-2009 (Dibber, 2009). These subunits were

used in current project for the penta-concatenation of GABAA ρ1 in order to obtain a tethered receptor of

identical subunits which could be site-specifically mutated. Dimers and trimers of ρ1 subunits tethered by six different linkers had additionally been synthesized by lab technician Lene G. Larsen prior to the onset of this project to enable determination of the optimal linker length as evaluated by functional expression

of the constructs in CHO cells by whole-cell patch clamp technology. The hetero-oligomeric GABAA dimer and trimer constructs were likewise a product of previous work by Lene G. Larsen.

______

3. EXPERIMENTAL PROCEDURES

The experimental procedures of this project have been divided into three parts:

Molecular biology: construction of GABAA ρ1 concatamers

Cell culture and transfection: expression of GABAA concatamers in Chinese Hamster Ovary Cells and Xenopus laevis oocytes

Electrophysiology: measuring the functional expression of GABAA concatamers using one- and two- electrode whole-cell patch clamp technology.

Plasmid maps are listed in Appendix B: Plasmids Kit-based protocols are listed in Appendix C: Protocols. Buffer compositions, media and chemicals are listed in Appendix D: Drugs and solutions.

Prior to this project, work in our lab generated the constructs presented in section 3.1.1-3.1.2. The strategy of the concatamer production was made by Marianne Lerbech Jensen. Defolliculation of Xenopus oocytes as described in section 3.2.4. was performed by lab technicians of Department of Receptor Pharmacology, NeuroSearch, as was the synthesis of the cRNA in table 3.11. Section 3.1.3 marks the onset of this project.

Concatenation of the Cys-loop receptors ______3.I MOLECULAR BIOLOGY

3.1.1 Subunits

GABAA ρ1 concatamers were assembled from five modified human ρ1 subunits termed ρ1A-E (GeneBank Acc. No. NM_002042). These subunits were identical in amino acid sequence but could be distinguished by the presence of two unique restriction sites in each subunit which had been introduced by site-directed mutagenesis. One site was created in the 5’ end of the ρ1 coding region and the other site was created in the 3’ end to enable identification and determination of subunit orientation in concatenated constructs. ρ1A-E were synthesized by a previous master student, Ditte Dibber (Dibber, 2009).

Subunit 5’ end unique site (1) 3’ end unique site (2) cDNA base pairs Vector

GABAA ρ1A A1: NheI A2: BsrGI 1422 pNS1zm*

GABAA ρ1B B1: SalI B2: PstI 1422 pNS1zm

GABAA ρ1C C1: BspEI C2: BsiWI 1422 pNS1zm

GABAA ρ1D D1: BstBI D2: SacII 1422 pNS1zm

GABAA ρ1E E1: AgeI E2: AclI 1422 pNS1zm

Table 3.1. Overview of unique restriction sites introduced into the GABAA ρ1 subunit isoforms A-E. *A NheI site was deleted from this vector.

Each subunit was subcloned into the pcDNA3-derived custom-made pNS1zm vector (appendix B6) which allows for high-level constitutive transcription in mammalian cells under control of the viral cytomegalovirus

(CMV) promoter. Sequences corresponding to the unique restriction sites in ρ1A-E were deleted from this vector before insertion of the subunits, however a NheI site (A1) located 68 base pairs upstream of the ρ1 coding region was forgotten. This site was subsequently removed from only pNS1zm-ρ1A, hence concatenation of ρ1A-E was performed in this plasmid.

3.1.2 Concatamers

A. GABAA ρ1

Dual and triple cDNA constructs were synthesized from a subset of the modified ρ1 subunits described in section 3.1.1. The subunit cDNAs were covalently linked by sequences that contained a varying number of amino acid residues, and used for the determination of optimal ρ1 linker length as described in section 3.3.6. Linker sequences were inserted to delete the stop codon after the coding region of the first subunit in a tandem, and to delete the signal sequence in the N-terminal of the second subunit in a tandem. A single reading frame was maintained to ensure proper transcription of the resulting fusion cDNA construct. The

Concatenation of the Cys-loop receptors ______

linker lengths in table 3.2 denote the length of the synthetic linker and do not include the C- and N-terminal protrusions of the tandem subunits.

Construct Linked subunits Amino acid sequence of linker Vector

L20 dimer ρ1B + ρ1C L20: Q5A2VPAQ2A3Q5 pNS1zm L20 trimer ρ1B + ρ1C + ρ1D

L23 dimer ρ1B + ρ1C L23: Q5A2VPAQ2A3PA2Q5 pNS1zm L23 trimer ρ1B + ρ1C + ρ1D

L25 dimer ρ1B + ρ1C L25: Q5A2VPAQ2A3PA2PAQ5 pNS1zm L25 trimer ρ1B + ρ1C + ρ1D

L30 dimer ρ1B + ρ1C L30: Q5A2VPAQ2A3 (PA2)2Q2A2Q5 pNS1zm

L40 dimer ρ1B + ρ1C L40: Q5A2VPAQ2A3 pNS1zm

L6ags dimer ρ1C + ρ1D L6ags: EG(AGS)6 pNS1zm

Table 3.2. Previously synthesized GABAA ρ1 concatamers used in the linker optimization experiment. “Lx” denotes “linker containing x amino acids residues”. “L6ags” denotes “linker containing 6 repeats of the amino acid sequence alanine-glycine-serine”. The first 8 linker sequences are derivations of the sequences used to link GABAA α1 and β2 subunits in Baumann et al..(2001). L6ags was copied from Zhou et al.. (2003). All concatamer constructs were subcloned in the pNS1zm vector.

B. GABAA α1β3γ2

Dual and triple GABAA α1β3γ2 cDNA constructs used for Xenopus oocyte patch clamp experiments were synthesized according to Baumann et al. (2001, 2002).

Construct Linked subunits Amino acid sequence of linker Vector

GABA β + α β3-α1 A 3 1 L23: Q3(Q2A3PA)2AQ5 pNS1z

γ2-β3-α1 GABAA γ2 + β3 +α1 L26: Q A PAQ (QA) A PA Q + pNS1z 5 3 2 2 2 2 5 L23: Q3(Q2A3PA)2AQ5

Table 3.3. Previously synthesized GABAA α1β3γ2 concatamers. “Lx” denotes “linker containing x amino acids residues”. pNS1z is a pcDNA3-derived custom-made vector similar to pNS1zm. The linkers were from Baumann et al.. (2001, 2002).

3.1.3 Overlap Extension Polymerase Chain Reaction Overlap extension is a two-step variant of the Polymerase Chain Reaction (PCR) technique, which can be applied to introduce mutations at distinct sites in a sequence or to splice genes. In the first step, overlapping complementary sequences are added to the 3’ ends of the two DNA fragments to be spliced, or to the DNA regions on both sides of the target sequence for mutation. This is achieved by the use of a set of end primers flanking the region to be modified, and a set of internal primers which have complementary “tail” overhangs

Concatenation of the Cys-loop receptors ______containing the junction sequence of the genes to be fused, or the mutation to be introduced. Secondly, the overlapping tails of the PCR products generated in the first step are allowed to hybridize, and the fusion product is elongated and amplified by addition of the flanking end primers (Heckman & Pease, 2007). Fig. 3.1 and 3.2 illustrate the first and second step of the process, respectively.

Primer Type DNA sequence

ρ1-L6ags s Tail sense 5'-CCG CGG GAA ATG CTG GAA GTG CTG GAA GTG CTG GAA GTA CTG AAA GCA GAA TGC AC-3'

ρ1-L6ags as Tail antisense 5'-TCC AGC ATT TCC CGC GGA CCC ACT GCC TGC ACC TTC GGA GAA AAT AGA CCA GTA TA-3'

308 as End antisense 5'-CAA ACT CTC CAC CTG CAC-3'

327 as End antisense 5'-TGT CAA CCT CTG AGA TGC-3'

935 s End sense 5'-CAC AAC GGT GCT GAC CAT G-3'

993 s End sense 5'-GTC TCC TAC ATC AAG GCC GTG-3'

Table 3.4. Primers used for overlap extension PCR reactions. Sequences corresponding to the linker tails are in bold. All primers were purchased from Eurofins MWG Synthesis GmbH, Ebersgberg, Germany. Primers were designed previously.

In this project overlap extension PCR was applied to synthesize the linker region connecting two subunits in a tandem construct. The Expand High Fidelity PCR System from Roche Applied Science was used for this purpose. The system combines the thermostable DNA polymerase Taq with the thermostable DNA polymerase Tgo, which possesses a proofreading 3’-5’ exonuclease activity to ensure the replication fidelity of the system.

Overlap extension PCR reaction: step one The first PCR reaction was performed to incorporate complementary linker sequences in the 3’ ends of two subunit cDNAs to enable subsequent splicing. This is exemplified with the amplification of ρ1A and ρ1B in figure 3.1. The C terminal region of ρ1A (AC) was amplified using the end primer 935s and the tail primer ρ1-

L6ags-as. The tail primer contained from 5’ → 3’ half of the linker sequence (overhang) followed by a sequence complementary to the 3’ coding region of ρ1 before the stop codon. The N-terminal region of ρ1B

(BN) was amplified using the end primer 308as and the tail primer ρ1-L6ags-s which contained from 5’ → 3’ the other half of the linker sequence (overhang) followed by a sequence complementary to the 5’ end coding region of ρ1 after the signal sequence. The resulting amplified ρ1A PCR fragment [AC-L6ags] contained from 5’

→ 3’ the C-terminal sequence of ρ1A including a unique recognition site for BsrGI and the first half of the linker whereas the amplified ρ1B PCR fragment [L6ags-BN] contained from 5’ → 3’ the second half of the linker followed by the N-terminal sequence of ρ1B including the unique recognition site for SalI.

Incorporation of the primers resulted in the removal of a stop codon in the ρ1A sequence and deletion of the signal sequence of ρ1B.

Concatenation of the Cys-loop receptors ______

Reaction 1a Reaction 1b

ρ1-L6ags as 308as AC BN 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ BsrGI SalI 935s ρ1-L6ags s

[AC-L6ags] [L6ags-BN] 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’

C terminal ρ1A region with L6ags L6ags N terminal ρ1B region with BsrGI site SalI site

Fig. 3.1. Primary overlap extension PCR reaction exemplified with templates ρ A and B. A denotes the C-terminal 1 C region of ρ1A whereas BN denotes the N-terminal region of ρ1B. Dark blue areas depict ρ1 sequences, yellow areas depict unique restriction sites, red areas depict the linker sequence and arrows depict primers.

This procedure was repeated to create the following pairs of linker constructs: [AC-L6ags] + [L6ags-BN], [BC-

L6ags] + [L6ags-CN], [CC-L6ags] + [L6ags-EN], [EC-L6ags] + [L6ags-DN]. Templates and primers used for these reactions are given in table 3.5.

Reaction Linker construct Template Primers Unique site

1a+b AC-L6ags hGABAA ρ1A 935s/993s + ρ1-L6ags-as BsrGI

2a+b L6ags-BN hGABAA ρ1 308 as/327as + ρ1-L6ags-s SalI

3a+b BC-L6ags hGABAA ρ1B 935s/993s + ρ1-L6ags-as PstI

4a+b L6ags-CN hGABAA ρ1C 308 as/327as + ρ1-L6ags-s BspEI

5a+b CC-L6ags hGABAA ρ1C 935s/993s + ρ1-L6ags-as BsiWI

6a+b L6ags-EN hGABAA ρ1E 308 as/327 as + ρ1-L6ags-s AgeI

7a+b EC-L6ags hGABAA ρ1E 935s/993s + ρ1-L6ags-as AclII

8a+b L6ags-DN hGABAA ρ1D 308 as/327as + ρ1-L6ags-s BstBI Table 3.5. Overlap extension PCR reaction step one.

Each template was incubated in two separate reactions (a+b) with two different sense or antisense end

primers, respectively. For example hGABAA ρ1A was incubated with 935s in one reaction and 993s in another

reaction, and hGABAA ρ1B was incubated with 308as in one reaction and 327as in another reaction. Additionally, for each reaction a negative control was made without template DNA (not shown).

Practically: 1 ng template was mixed with 1X Expand High Fidelity buffer supplemented with 1.5 mM

MgCl2, 200 µM of each dNTP, 0.5 μM sense primer and 0.5 µM antisense primer, 0.9 units of ExpHF enzyme mix and DEPC water was added to a final volume of 50 µl. Template was replaced by DEPC water in the

Concatenation of the Cys-loop receptors ______negative controls. The reaction mixture was coated with a droplet of mineral oil to prevent evaporation. The PCR reaction was performed in an automated Stratagene Robocycler 40 (Agilent Technologies, Cedar Creek, Texas, USA) programmed as follows:

Initiation 3 minutes 94°C 1 cycle Denaturation 1 minute 94°C Annealing 1 minute 55°C 25 cycles Elongation 1 minute 72°C Final elongation 3 minutes 72°C 1 cycle

Overlap extension PCR: step two Each pair of linker constructs were subjected to another overlap extension PCR reaction to hybridize the 3’ end overlapping tails. For example [AC-L6ags] was incubated with [L6ags-BN] to allow annealing and elongation of the L6ags complementary sequences as shown in fig. 3.2, and the ρ1- specific end primers were added subsequently to amplify the resulting [AC-L6ags-BN]. Primers and templates for generation of the linker regions

[AC-L6ags-BN], [BC-L6ags-CN], [CC-L6ags-EN] and [EC-L6ags-DN] are summarized in table 3.6.

Fig. 3.2. Secondary overlap [AC-L6ags] extension PCR reaction [L6ags-BN] 5’ 3’ exemplified with [A1-L6ags] BsrGI 3’ 5’ SalI and [L6ags-B1]. Dark blue areas depict ρ1 sequences, yellow areas depict unique [AC-L6ags] [L6ags-BN] 3’ restriction sites, red areas 5’ 5’ 3’ depict the linker sequence and arrows depict primers. The Addition of end primers after linker constructs hybridize by 10 cycles their complementary tail 308as overhangs and elongate. [AC-L6ags-BN] 5’ 3’ Amplification is achieved by

3’ 5’ addition of ρ1 end primers after 10 cycles of PCR. 935s

Reaction Linker region Template pairs Primers Unique sites

1 [AC-L6ags-BN] [AC-L6ags] + [L6ags-BN] 935 s + 308 as BsrGI + SalI

2 [BC-L6ags-CN] [BC-L6ags] + [L6ags-CN] 935 s + 308 as PstI + BspEI

3 [CC-L6ags-EN] [CC-L6ags] + [L6ags-EN] 993 s + 327 as BsiWI + AgeI

4 [EC-L6ags-DN] [EC-L6ags] + [L6ags-DN] 993 s + 308 as AclII + BstBI Table 3.6. Overlap extension PCR reaction: step two. 8 linker constructs were chosen from the primary overlap-extension PCR reaction and incubated in pairs of two to generate 4 linker regions.

37 ers.

Concatenation of the Cys-loop receptors ______

Practically: for each of the 4 reactions 2 µl of each template was mixed with 1XRB buffer supplemented with

1.5 mM MgCl2, 200 µM of each dNTP, 0.9 units of ExpHF and 35,75 µl DEPC water, and one droplet of mineral oil was added to the PCR mixture.

Initiation 3 minutes 94°C 1 cycle Denaturation 1 minute 94°C Annealing 1 minute 55°C 30 cycles Elongation 2 minutes 72°C Final elongation 3 minutes 72°C 1 cycle

The first 10 cycles of PCR were performed without end primers. In these cycles the PCR fragments served as both template and primer for each other because of the overlapping tail sequences. 0.5 μM of ρ1 sense and antisense end primer was added to the PCR reaction subsequently. All PCR products were visualized on a 1.2 % agarose gel to verify the presence and size of the constructs.

3.1.4 Purification of PCR products and ligation fragments PCR products were purified according to the PCR clean-up Nucleospin® Extract II protocol from Macherey- Nagel (appendix C1). The protocol is based on the principle that DNA molecules > 100 base pairs adsorb to a silica membrane in the presence of chaotropic salt, whereas PCR contaminants such as primer DNA, enzymes, unincorporated nucleotides and oils can be washed out by addition of ethanol-containing buffer followed by centrifugation.

Ligation fragments were purified by gel extraction using the same kit following the procedure described below.

Practically: 10 µg DNA was mixed with 2 µl loading buffer and placed in the large lane of a 1.2% agarose gel. In the neighbouring lane 0.5 µg of the same DNA was loaded as reference alongside a 1 kb Molecular Size marker. After voltage-applied incubation for approximately 30 minutes at 80 V (depending on the fragment size) the lane containing 10 µg DNA was separated from the rest of the gel with a scalpel and the reference lane was visualized by UV light together with the 1 kb marker lane. The position of the relevant DNA fragment was marked on the reference lane, and the marking was used to estimate the position of the 10 µg fragment in the parallel non-visualized lane. The fragment was excised with a scalpel and treated according to the Macherey-Nagel NucleoSpin® Extract II Protocol for DNA extraction from agarose gels is described in appendix C2.

Concatenation of the Cys-loop receptors ______

3.1.5 Agarose gel electrophoresis PCR products and restriction enzyme digested DNA fragments were separated according to size via agarose gel electrophoresis.

Practically: gels were prepared in a UV-transparent gel tray with a fixed height comb and contained 1.2% agarose in TBE buffer and 0.5 µg/ml ethidium bromide. 0.5 µg DNA was mixed with 2 µl loading buffer prior to loading alongside a 1 Kb molecular weight marker (New England Biolabs, cat# 10787-018) for determination of fragment size. Gels were placed in a Bio-Rad Mini-Sub® Cell GT Base (cat# 170-4486) at 60 -100 V for 15-90 minutes and DNA was visualized by exposure to UV light at 302 nm and photographed. For gel extraction purposes, the DNA to be excised was not photographed due to the mutagenic effect of UV light.

3.1.6 Determination of DNA concentration The concentration of nucleic acids was determined by UV spectrophotometry.

Practically: 1-2 µl DNA or RNA solution was diluted to 100 µl with nuclease-free water and transferred to an Eppendorf UVette® (cat# 0030 106.318) which was placed in a biophotometer (Eppendorf AG, Hamburg,

Germany). The absorbance at 260 nm (A260) was measured and the concentration was calculated by the formula:

[DNA] = A260 · 50 µg/ml · D or [RNA] = A260 · 40 µg/ml · D

D is the dilution factor. Prior to measurement of a sample value the blank for an UVette® containing 100 µl nuclease-free water was determined.

3.1.7 TOPO cloning Subcloning of the PCR fusion products into the TOPO plasmid vector was done using the Zero Blunt® TOPO® PCR Cloning Kit from Invitrogen (appendix C5). This kit provides a linearized plasmid TOPO vector with Vaccinia virus DNA topoisomerase I covalently bound to the phosphate group at the 3’ end of each DNA strand. The vector-topoisomerase I-complex mediates phosphate linkage between the 3’ ends of the vector and the 5’ ends of blunt or cohesive PCR fragments thus ligating the insert into the vector. The TOPO® cloning reaction was transformed into chemically competent E. coli One Shot® TOP10 cells (cat# C4040-03, Invitrogen).

Practically: transformed bacteria were cultured at 37°C for one hour in rich non-selective medium to allow the expression of a zeocin resistance gene conferred to the TOPO vector. Subsequently they were spread on prewarmed zeocin-selective Luria bertani (LB) agar plates (25 µg/ml) and incubated at 37°C overnight. The

Concatenation of the Cys-loop receptors ______following day 12 individual colonies were picked from the plates and transferred to falcon-tubes containing 1.5 ml LB media supplemented with zeocin (25 µg/ml). These cultures were then incubated at 37°C with vigorous shaking (280 rpm) overnight and plasmid DNA purification was performed the next day according to section 3.1.8.

TOPO clones were analyzed by restriction analysis with EcoRI which cleaves the plasmid DNA 11 and 7 base pairs before and after the insert respectively, and with the unique restriction enzymes that cleave the PCR insert in the 5’ and 3’ end. 2-3 clones were chosen for ethanol precipitation and sequencing.

3.1.8 Isolation of plasmid DNA from Bacterial Colonies The Promega Wizard® Plus SV Minipreps DNA Purification System (appendix C3) was used to purify plasmid DNA from small-scale bacterial cultures of 1-10 ml. The kit takes advantage of the differential denaturation and renaturation characteristics of plasmid DNA versus chromosomal DNA and is based on alkaline lysis of bacteria followed by adsorption of DNA onto a silica based membrane column in the presence of chaotropic salts. The procedure consists of the following steps:

1. Alkaline lysis of the bacteria in the presence of SDS and proteases results in the denaturation and degradation of proteins. Chromosomal DNA breaks into linear fragments whereas plasmid DNA remains circular and interlocked. 2. Neutralization of the lysate is achieved with a solution containing guanidine hydrochloride and potassium acetate. Guanidine hydrochloride is a chaotropic salt which acts as a denaturant and promotes the dehydration and binding of DNA onto a silica membrane. Potassium acetate promotes the precipitation of high-molecular weight substances such as bacterial membrane components, proteins and chromosomal DNA in the presence of SDS. Upon neutralization interlocked plasmid DNA strands rehybridize and remain in solution whereas the chromosomal DNA fragments form insoluble aggregates. 3. Separation of precipitate and cleared lysate is followed by adsorption of plasmid DNA onto a silica-based membrane column. 4. Washing of the membrane with ethanol-containing buffer removes remaining salts and impurities. 5. Elution of plasmid DNA is achieved with a low-ionic-strength slightly alkaline buffer.

For scale-up plasmid isolation from bacterial cultures of 50 ml the Qiagen HiSpeed Plasmid Purification Midi Kit was used (appendix C4). This kit is based on the same alkaline lysis principle as the Promega Wizard® Plus SV Minipreps DNA Purification System but isolates plasmid DNA by anion exchange chromatography instead of silica-gel-membrane technology.

Concatenation of the Cys-loop receptors ______

3.1.9 Restriction enzymes Restriction enzyme analysis was performed to verify the insertion and orientation of DNA fragments after subcloning and ligation.

Practically: In general 0.5 µg DNA was mixed with 1 µl of the recommended NeBuffer, 1 µl 10X BSA (if required) and 0.1 µl enzyme. DEPC water was added for a final volume of 10 µl and the mixture was incubated at the relevant temperature for one hour. If a double digestion was performed using restriction enzymes with different optimum temperatures, the enzyme with the lowest optimum was added first and after 45 minutes of incubation the second enzyme was added and the temperature was elevated. Digestion products were analysed via agarose gel electrophoresis as described in section 3.1.5.

Enzyme Recognition site Units/ml BSA Buffer Temp. °C Cat #

AclI 5'…AA↓CGTT…3' 3.000 + NeBuffer 4 37 R0598L

AgeI 5'…A↓CCGGT…3' 5.000 - NeBuffer 1 37 R0552L

BsiWI 5'…C↓GTACG…3' 10.000 - NeBuffer 1-3 55 R0553L

BspEI 5'…T↓CCGGA…3' 10.000 - NeBuffer 3 37 R0540L

BsrGI 5'…T↓GTACA…3' 10.000 + NeBuffer 2, 4 37 R0575L

BstBI 5'…TT↓CGAA…3' 20.000 - NeBuffer 4 65 R0519L

EcoRI 5'…G↓AATTC…3' 20.000 - NeBuffer 1-4 37 R0101L

NheI 5'…G↓CTAGC…3' 10.000 + NeBuffer 1, 2, 4 37 R0131L

PstI 5'…CTGCA↓G…3' 20.000 + NeBuffer 3 37 R0140L

SacII 5'…CCGC↓GG…3' 20.000 - NeBuffer 4 37 R0157L

SalI 5'…G↓TCGAC…3' 20.000 - NeBuffer 3 37 R0138L

SphI 5'…GCATG↓C…3' 10.000 - NeBuffer 1, 2, 4 37 R0182L

XbaI 5'…T↓CTAGA…3' 20.000 + NeBuffer 2, 4 37 R0145L

XhoI 5'…C↓TCGAG…3' 20.000 + NeBuffer 2-4 37 R0146L

Table 3.7. Restriction endonucleases used for the concatenation of GABAA ρ1A-E. BSA = Bovine Serum Albumin. Enzymes, buffers and BSA were New England Biolabs®Inc products purchased from Medinova Scientific A/S, Glostrup, Denmark.

3.1.10 Ethanol precipitation PCR samples were precipitated by addition of sodium acetate and ethanol.

Practically: DNA was ethanol precipitated by addition of 0.1 x 3M sodium acetate and 2.5 x 96% chilled ethanol. The solution was incubated at -20°C for 15 minutes and centrifuged at 20.000 g for 20 minutes at 4°C. The supernatant was carefully discarded and the pellet was rinsed with 70% chilled ethanol. The sample

Concatenation of the Cys-loop receptors ______was spun again at 20.000 g for 5 minutes at 4°C and the supernatant was discarded. The pellet was air-dried for 5 minutes at room temperature and resuspended in TE buffer for a final concentration of 1 µg/ul.

3.1.11 Sequencing TOPO cloned PCR fragments were sequenced at MWG Biotech AG, Ebersberg, Germany.

Sequences were checked by alignment with the linker sequence and the relevant GABAA ρ1 A-E sequences using the software programs Seqman and SeqBuilder from DNASTAR* Lasergene (Madison, WI, USA).

3.1.12 Retransformation Verified PCR fusion constructs were retransformed into electrocompetent E. coli XL1-Blue MRF’ cells (Stratagene, cat# 200158) by electroporation.

Practically: 50-100 pg plasmid was added to 40 µL XL1-Blue cells thawed on ice and transferred to a chilled Apollo 2 mm electroporation cuvette which was kept on ice. Samples were pulsed once in a Bio-Rad MicroPulser™ Electroporator with 2.5 kilovolts and 1 ml LB media was added immediately. Cells were incubated for one hour at 37°C with vigorous shaking to allow expression of the TOPO zeocin-resistance gene and were subsequently plated on zeocin-selective LB plates. Plates were incubated at 37°C overnight and two colonies were transferred to a 500 ml flask containing 50 ml LB medium + zeocin (25 µg/ml). The cultures were incubated at 37°C overnight with vigorous shaking. Amplified clones were purified with the Qiagen HiSpeed Plasmid Purification Midi Kit (appendix C4) and the concentration was adjusted to 1 µg/ul by ethanol precipitation and resuspension in TE buffer.

3.1.13 Ligation Two ligation reactions were performed in series for the concatenation of each subunit: first, the subunit cDNAs to be tethered were ligated into the same vector, and secondly the PCR linker region was inserted between the two sequences. The strategy is outlined in figure 3.3. T4 DNA ligase purchased from New England Biolabs (Medinova Scientific A/S, Glostrup, Denmark) was used for all ligation reactions. This enzyme catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA, and joins blunt or cohesive DNA fragments.

Ligation of subunits: step one

Linearization: the pNS1zm ρ1 vector was linearized with XbaI which cleaves the plasmid 11 basepairs

after the coding sequence of ρ1 and purified on a column according to the PCR clean-up Nucleospin® Extract II protocol from Macherey-Nagel (appendix B1). Subsequently it was dephosphorylated with

Concatenation of the Cys-loop receptors ______

Shrimp Alkaline Phosphatase (SAP) from USB Affymetrix (cat# 70092Y) according to manufacturer’s instruction (appendix C6) to prevent religation.

Excision: the ρ1 subunit to be inserted was excised from pNS1zm with SpeI and XbaI, which cleave at

recognition sequences located 28 and 11 basepairs before and after the coding sequence of ρ1, respectively, and purified according to the Macherey-Nagel PCR clean-up Nucleospin® Extract II protocol for DNA extraction from agarose gels (appendix C2). Ligation: ligation was performed at a molar ratio of approximately 1:3 between vector and fragment according to a formula that takes into account the relative length of the insert and the vector:

ng fragment = 3 x (ng vector/( basepairs in vector /basepairs in fragment))

A ligation mix was prepared of 50 ng vector, the calculated amount of fragment, 1X T4 buffer and approximately 400 units of T4 DNA ligase enzyme. DEPC water was added to a final volume of 10 µl. An additional control reaction was made with DEPC water replacing fragment to check the efficiency of the SAP reaction. The reactions were incubated at 12°C overnight and the following day the T4 ligase was inactivated at 65°C for 20 minutes. Transformation: the ligations were electroporated into E. coli XL1-Blue cells and plated as described in section 3.1.7. The colony number was determined the following day, and if the ratio of ligation to control was less than 10:1 SAP treatment of the vector and ligation was repeated. 12 clones were amplified as described in section 3.1.7.

Insertion of linker region: step two Linearization: the pNS1zm vector carrying the tandem subunits was digested with two unique restriction enzymes to excise the region between the 3’ end of the first subunit cDNA and the 5’ end of the second subunit cDNA, and the linearized vector was purified by agarose gel extraction. Excision: the PCR linker region was excised from the TOPO vector by way of the two unique restriction sites, and the fragment was purified as described in section 3.1.4. Ligation: vector and fragment were incubated in a ligation reaction as described above. The resulting

pNS1zm ρ1 tandem with L6ags linking the subunits represented the end product of one ligation series.

Table 3.8 summarizes the ligation reactions performed to obtain the ρ1 L6ags concatenated pentamer. Briefly, subunits A and B were linked in parallel to insertion of the previously synthesized dimer L6ags C-D into pNS1zm ρ1B. Subsequently L6ags B-C-D was ligated into L6ags A-B to generate L6ags A-B-C-D. The presence of a SacII site in both the D subunit and L6ags made it impossible to insert a linker region between DC and EN in a pentamer intermediate L6ags A-B-C-DE. Therefore, the D subunit was removed from L6ags A-B-C-D and subunit E plus the linker region [CC-L6ags-EN] were inserted downstream of L6ags A-B-C via three-way ligation.

Concatenation of the Cys-loop receptors ______

Subunit D was ligated into the resulting pNS1zm L6ags A-B-C-E tetrameric construct and finally the linker region [EC-L6ags-DN] was inserted between subunit E and D creating a final L6ags pentamer with the subunit sequence A-B-C-E-D. Curiously three-way ligation only worked for the synthesis of L6ags A-B-C-E. Therefore, two-way ligation was performed for all other reactions.

Construct Step Vector linearization Fragment excision Ligation product

Dimer 1 pNS1zm ρ1A // pNS1zm ρ1B// pNS1zm XbaI XbaI + SpeI ρ1AB

2 pNS1zm ρ1AB // pTOPO [AC-L6ags-BN] // pNS1zm BsrGI + SalI BsrGI + SalI ρ1 L6ags A-B

Trimer 3 pNS1zm ρ1B // pNS1zm ρ1 L6ags C-D* // pNS1zm XbaI XbaI + SpeI ρ1L6ags BC-D

4 pNS1zm ρ1BC-L6ags-D // pTOPO [BC-L6ags-CN] // pNS1zm PstI + BspEI PstI + BspEI ρ1 L6ags B-C-D

Tetramer 5 pNS1zm ρ1 L6ags A-B// pNS1zm ρ1 L6ags B-C-D // pNS1zm PstI + XbaI PstI + XbaI ρ1 L6ags A-B-C-D

6 pNS1zm ρ1 L6ags A-B-C-D // pTOPO [CC-L6ags-EN] // BsiWI + AgeI pNS1zm BsiWI + XbaI pNS1zm ρ1E // AgeI + XbaI ** ρ1 L6ags A-B-C-E

Pentamer 7 pNS1zm ρ1 L6ags A-B-C-E // pNS1zm ρ1D // pNS1zm XbaI XbaI + SpeI ρ1 L6ags A-B-C-ED

8 pNS1zm ρ1 L6ags A-B-C-ED // pTOPO [EC-L6ags-DN] // pNS1zm AclI + BstBI AclI + BstBI ρ1 L6ags A-B-C-E-D

Table 3.8. Concatenation of ρ1 subunit was performed in two ligation steps for each construct. // denotes “digested with”. * This dimer pNS1zm ρ1 L6ags C-D was made previously. ** This step was a three-way ligation, hence two fragments were inserted into the linearized vector.

3.1.1 4 Verfication of ligation products For subunit ligation, presence and orientation of the insert were initially confirmed by cleaving a unique recognition site in the insert and a recognition site in the vector. Subsequently, 2-3 clones were chosen for further amplification as described in section 3.1.12 and final verification was performed with exhaustive restriction enzyme analysis. Correct insertion of the linker region deleted a XhoI site located between the coding sequences of the concatenated subunits, and introduced a SacII site instead. Presence of the linker region was therefore verified by restriction digestion with XhoI and SacII. The integrity of the unique restriction enzyme recognition sites after insertion of the linker region was verified additionally. Digestions are presented in section 4: Results.

Concatenation of the Cys-loop receptors ______

Concatenation

A. Subunit ligation

A B A XbaI SpeI XbaI

pNS1zm pNS1zm pNS1zm

ρ1A ρ1B ρ1A

1. 2. 3.

1. Linearization of pNS1zm ρ1A with XbaI followed by column purification and treatment with shrimp alkaline phosphatase

2. Excision of ρ1B from vector with XbaI and SpeI followed by gel extraction and purification 3. Ligation of ρ1B excised fragment into linearized pNS1zm-ρ1A followed by E. coli transformation, selection, amplification and RE verification of ligated construct

B. Insertion of linker region

BsrGI SalI BsrGI SalI A B A B pNS1zm pTopo pNS1zm [A -L -B ] ρ1AB C 6ags N ρ1AB

4. 5. 6.

4. Linearization of pNS1zm ρ1AB with BsrGI and SalI followed by gel purification 5. Excision of [AC-L6ags-BN] linker region from vector with BsrGI and SalI followed by gel extraction and purification

6. Ligation of excised [AC-L6ags-BN] linker region into linearized pNS1zm-ρ1AB followed by E. coli transformation, selection, amplification and RE verification of ligated construct

A B

pNS1zm

L6ags ρ1A-B

Fig. 3.3. Flowchart outlining the concatenation strategy for the synthesis of GABAA ρ1 L6ags concatamers. Dark blue regions represent ρ1 sequences, yellow regions represent unique restriction sites, red regions represent the sequence of L6ags and arrows represent restriction enzymes.

Concatenation of the Cys-loop receptors ______

3.1.15 cRNA synthesis and purification cRNA was prepared using the Ambion mMessage mMachine T7 Transcription Kit Protocol (appendix C7). This kit enables transcription of linearized template cDNA that contains a T7 RNA polymerase promoter site. T7 RNA polymerase catalyzes the transcription of DNA sequences downstream of the promoter site and incorporates the cap analog [m7G(5’)ppp(5’)G] as the 5’ terminal G of the transcript.

Practically: 10 ug cDNA of each construct was linearized with XbaI which cleaves the pNS1zm vector 11 basepairs downstream of the insert. Because transcription proceeds to the end of the template, linearization of the plasmid DNA ensures RNA transcripts of a defined length and sequence. 1 µl of the eluate was visualized on a gel to confirm complete linearization. The DNA concentration was determined as described in section 3.1.6. The reaction mixture was treated with Proteinase K to remove potential contaminants such as RNases and transcription inhibitors and DNA was purified using the Qiagen Nucleospin Extract II DNA purification protocol as described in appendix C4. Finally, a 6X transcription and capping reaction was made according to appendix C7.

The cRNA was purified using the Qiagen RNeasy Mini Kit RNA Cleanup protocol (appendix C8). The principle behind this protocol is similar to the one described in section 3.1.4. The cRNA was eluted in nuclease-free water and the concentration was measured as described in section 3.1.6. The final concentration was adjusted to 1 µg/µl with nuclease-free water and the cRNA was stored at -80 °C.

3.2 CELL CULTURE AND TRANSFECTION

3.2.1 Chinese Hamster Ovary Cells (CHO cells) CHO-K1 (ATCC no. CCL-61) is a subclone of the mammalian Chinese Hamster Ovary cell line. This cell line was chosen for the expression of GABAA ρ1 concatenated constructs because it is characterized by rapid growth and it yields high expression of the wild type GABAA ρ1 receptor upon transient transfection.

Practically: CHO-K1 cells were maintained in Dulbecco’s Modified Eagle Medium (D-MEM) with 25 mM D-glucose, 1 mM sodium pyruvate and 3.97 mM glutamax (cat# 31966-021 GIBCO®, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-proline. The cells were cultured as a 2 monolayer in 75 cm cell tissue flasks at 37 °C in a humidified atmosphere of 5% CO2 and 95% air and passaged 3 times a week.

Concatenation of the Cys-loop receptors ______

3.2.2 Transient transfections

The pNS1zm-ρ1 concatamers were expressed in CHO cells together with the plasmids pNS1z-EGFP and a 1:1 mixture of pNS1z-ASIC1A and pNS1z-ASIC3. pNS1z-EGFP encodes the Enhanced Green Fluorescent Protein which functioned as a reporter of expression for the identification of successfully transfected cells. pNS1z + ASIC1A+3 encode an Acid-Sensing Ion-Channel which conducts predominantly Na ions in response to an + elevated concentration of [H ]. The pNS1z ASIC1A+3 were co-transfected for verification of the patch clamp application system.

CHO cells at 80-90% confluence were transiently transfected with the relevant pNSz1m-ρ1 mixture in a serum free medium using the LipofectAMINE PLUSTM kit from Invitrogen. The principle behind this kit is a lipid- based transfection technology in which vesicles of cationic lipids interact spontaneously with negatively charged plasmid DNA to form lipid-DNA complexes. As the negative charge of the nucleic acid is masked by the surrounding cationic lipid the complex is allowed to fuse efficiently with the plasma membrane of cells resulting in both uptake and expression of DNA (Felgner et al., 1994; Felgner et al., 1987).

Practically: 6 µl of Lipofectamine™ LTX reagent and 4 µl PLUS™ Reagent were each diluted with 125 µl OptiMEM®I Reduced Serum Medium in separate vials. 1 µl purified plasmid DNA was mixed with the diluted PLUS™ Reagent and the two reactions were incubated for 15 minutes at room temperature. Next, the vials were mixed and incubated for 15 minutes at room temperature. The transfection mixture was added to cells which had been seeded in 6-well plates and covered with 500 µl OptiMEM®I Reduced Serum Medium. The cells were incubated at 37°C for 3 hours and finally the medium was changed to FBS- and L-proline supplemented D-MEM.

DNA mixture pNS1zm ρ1 construct pNS1z EGFP pNS1z ASIC1A+3 1:1

Monomer 0.8 µg 0.1 µg 0.1 µg

Dimer 0.8 µg 0.1 µg 0.1 µg

Trimer 0.8 µg 0.1 µg 0.1 µg

Dimer + trimer 1:1 0.4 µg + 0.4 µg 0.1 µg 0.1 µg

Tetramer 0.8 µg 0.1 µg 0.1 µg

Pentamer 0.8 µg 0.1 µg 0.1 µg 0.9 µg 0.1 µg 0.0 µg 1.8 µg 0.1 µg 0.1 µg 0.4 µg 0.05 µg 0.05 µg

Table 3.9. Plasmid μg ratios for CHO transfection mixtures.

Concatenation of the Cys-loop receptors ______

0.1 µg pNS1z-EGFP, 0.1 µg pNS1z-ASIC1A+3 1:1 and 0.8 µg pNS1zm containing the relevant concatenated

GABAA ρ1 construct were mixed for a final transfection amount of 1 µg DNA. Dimer and trimer were co- transfected in a 1:1 ratio yielding 0.4 µg of each construct per transfection. The pentamer was additionally transfected without ASIC1a+3 or with a half or double amount of DNA in attempt to increase expression.

Transfected cells were allowed to grow in FBS- and L-prolin-supplemented D-MEM for ~20 hours to allow expression of the DNA constructs. Cells were then washed once with sterile PBS, incubated with Trypsin- EDTA for 1-3 minutes at 37 °C followed by resuspension and dilution in D-MEM. Finally, cells were seeded onto 3.5 mm poly-d-lysine- coated glass coverslips for patch clamp experiments which were performed on the same or the following day.

3.2.3 Xenopus laevis oocytes

Oocytes from the South African clawed frog Xenopus laevis were chosen for the expression of GABAA α1β3γ2 concatamers. Xenopus oocytes have a large diameter of approximately 1 mm which facilitates cRNA injection and electrophysiological experiments. Furthermore, they are characterized by a high translation level of heterologous cRNA and by efficient insertion of functional mammalian membrane proteins into their plasma membranes (Tammaro et al., 2008). Oocyte positive laboratory-bred Xenopus laevis females were from Nasco (Modesto, CA, USA, cat# LM00716MX).

3.2.4 Defolliculation Three to four lobes were surgically removed from the ovaries of adult female Xenopus laevis wich had been anaesthesized by hypothermia and immersion into a solution containing tricaine (2.8 g to 1 liter of water). The lobes were manually dissected with a pair of scissors, and the oocytes (stage V-VI) were washed 5-6 times with Low Calcium Barth’s solution (LBA). Defolliculation was achieved with collagenase type 1A diluted to 3 mg/ml in LBA for 60-90 minutes at 18 degrees under constant agitation. Next, the oocytes were washed 5-6 times in Modified Barth’s solution (MBA) and sorted manually under a stereomicroscope. They were kept at 4°C in fresh MBA at until microinjection.

3.2.5 Microinjection Micropipettes for cRNA injection were prepared from fire-polished borosilicate filament tubes (Sutter Instruments, Novato, CA, USA, cat# BF100-50-10) and fabricated on a DMZ-universal puller (Zeitz Instrumente, Bayern, Germany). They were placed in the pipette holder of a manual micromanipulator and the most distal part of the tip was manually removed with tweezers to increase the opening diameter. The pipette tip was submerged in nuclease-free water and suction was applied by activating a pneumatic PV840 picopump (World Precision Instruments, Sarasota, FL, USA) connected to the pipette holder. The

Concatenation of the Cys-loop receptors ______micropipette was then calibrated by injecting a droplet of water into mineral oil under a stereo microscope and adjusting the output until a droplet diameter corresponding to a volume of 40 nl was produced per ejection. The remaining nuclease-free water was ejected from the pipette and the relevant cRNA was drawn up by vacuum. Oocytes covered in MBA were placed row-wise in a grooved injection chamber and 40 nl cRNA was injected into each egg by positive pressure. Finally, injected eggs were transferred to a petri dish, sorted, washed and incubated in MBA at 18 degrees for 2-3 days.

Table 3.10. cRNA ratios for Xenopus oocyte cRNA mixture Subunit Concentration microinjections. For the GABAA α1β3γ2 concatamers cRNA solutions were prepared with α1+β3+γ2 WT 1:1:2 α1 0.125 µg/µl a final concentration of 0.25-0.50 µg/µl. β3 0.125 µg/µl γ2 0.250 µg/µl

α1+β3 WT 1:1 α1 0.125 µg/µl β3 0.125 µg/µl

β3-α1 β3-α1 0.250 µg/µl

β3 -α1 + γ2 1:1 β3-α1 0.250 µg/µl γ2 0.250 µg/µl

γ2-β3-α1 γ2- β3 -α1 0.250 µg/µl

β3-α1 + γ2- β3-α11:1 β3-α1 0.250 µg/µl γ2- β3 -α1 0.250 µg/µl

3.3 ELECTROPHYSIOLOGY

Patch clamp technology was applied for the functional analysis of GABAA concatamers. Initially, GABAA ρ1 dimer and trimer constructs with different linkers were screened for functional expression in CHO cells. Next, synthesized GABAA ρ1 L6ags concatamers were tested for functional expression in CHO cells and finally,

GABAA α1β3γ2 concatamers were analyzed with two-electrode whole-cell voltage clamp in oocytes.

3.3.1 The Patch Clamp Technique Patch clamp is a high-resolution electrophysiological method which allows the delicate measurement of transmembrane ionic currents and the study of single or multiple ion channels in the plasma membranes of living cells. It was developed by Bert Sakmann and Erwin Neher in 1976, and revolutionized the study of ion channels because it enabled the direct observation of channel kinetics and conductance via single-channel recordings (Hille, 2001). Patch clamp recordings begin with the formation of a contact between the open tip of a blunt micropipette and the surface of a cell. The cell is placed in a recording chamber which contains a reference electrode, and is continually superfused with ionic solution. The pipette is filled with ionic solution,

Concatenation of the Cys-loop receptors ______and contains a recording electrode. Both electrodes are coupled to an amplifier which enables monitoring and changing of ionic fluxes and potential differences between them (Molleman, 2003). The ionic composition of the pipette and bath solutions may resemble the intracellular and extracellular milieu of the cell and can be varied according to the desired patch clamp configuration as described in figure 3.5. Following contact between the pipette and the cell, negative pressure is applied to the pipette until a tight seal of >1GΩ resistance has been created between the membrane and the pipette tip. The high resistance and stability of this seal minimizes the current loss through the pipette/membrane interface and makes it possible to record single- channel or macroscopic whole cell currents over the membrane via the electrodes. Furthermore, it enables the electrical and mechanical isolation of the pipette-covered patch (Molleman, 2003).

Once the gigaseal has been established, 5 different patch clamp configurations can be obtained as illustrated in figure 3.4. Only the whole-cell configuration was applied in the presented CHO cell experiments.

3.3.2 Voltage clamp Voltage clamp involves the measurement of currents passing through the cell membrane at a clamped, constant membrane potential. A rapid electronic feedback system continually monitors the membrane potential and compares it to a holding potential set by the experimenter. Whenever the recorded potential deviates from the set potential a compensatory current will be instantly injected into the cell to cancel the difference. This compensatory current will be equal in amplitude and waveform to the current flowing through the ion channels of the membrane, but opposite in sign (Molleman, 2003). In a voltage-clamp experiment where the number of open channels is denoted N and the conductance of each channel is denoted γ, the total conductance is equal to the product, N ∙ γ. The electrochemical gradient of the permeable ion species, ΔV, produces a current of the magnitude, I = N ∙ γ ∙ ΔV. From this equation it is evident that the current generated in voltage clamp experiments is directly proportional to the number of open channels in the plasma membrane and thus reflects changes in ion channel activity (Sherman-Gold, 2008). In single-electrode voltage clamp (SEVC) measurements, recording of the membrane potential and injection of compensatory current is performed by only one electrode, whereas in two-electrode voltage clamp (TEVC) there is an electrode for each of these functions.

3.3.3 Errors of voltage clamp

A. Series resistance and cell capacitance The sum of the pipette resistance and the resistance of the residual ruptured patch under the pipette is called the series resistance (Rs). A high series resistance will cause a significant voltage drop over the pipette

Concatenation of the Cys-loop receptors ______

Patch clamp configurations

Apply negative pressure to obtain a gigaseal of > 1 GΩ

1. Cell attached configuration Allows the study of single channel activity in the pipette-covered patch of the intact cell. This configuration does not allow manipulation of the intracellular milieu and no direct measurement of the membrane potential can be made. However, in contrast to the other configurations it leaves the cell intact.

Apply a brief pulse of suction to rupture the membrane patch covered by the Nystatin or amphotericin B in the Withdraw the pipette from the cell to pipette tip pipette will decrease the resistance pull out a patch of membrane which of the sealed patch to selected small will spontaneously form a vesicle ions

2a. whole cell configuration Allows recording of macrocurrents flowing through all ion channels of the cell membrane. The position of an electrode on either side of the membrane enables the direct recording of the membrane potential. It is possible to manipulate Vesicle formation the intracellular milieu of the cell since the tip of the open pipette is big enough to allow mixing of the cytoplasm and the pipette solution. 2b. Perforated configuration Briefly lift the pipette above the However, this involves the risk of washing out of Allows the measurement of whole-cell bath. Upon exposure to air the cytosolic factors relevant to the ion channel macrocurrents while preserving the vesicle is destroyed, leaving a patch intracellular milieu of the cell. under study (ATP, Mg2+, PKA catalytic subunit) with the cytosolic side facing the The perforated patch configuration is associated with the disadvantage of bath compromized electrical access to the cell and Withdraw the requires controlled application of the pipette from the cell perforating drug. to pull out a patch of membrane which spontaneously reseals into a patch covering the pipette

3. Outside-out configuration 4. Inside-out configuration Outside-out patches are useful when studying This technique is suitable for the study of extracellular single channel regulation because it intracellular modulation of ion channel is possible to control the composition of both function at the single channel level. Like intracellular and extracellular solution. However, outside-out patches, this configuration there is a risk of losing important unknown involves washout of cytosolic factors and cytosolic factors, and moreover the cytoskeletal disruption of cytoskeletal cell structures. structures of the cell are disrupted when the patch is pulled.

Fig. 3.4. Flowchart depicting 5 different patch clamp configurations. Text reference: (Molleman, 2003).

Concatenation of the Cys-loop receptors ______and the ruptured patch when large currents are measured, and generate a deviation between the applied command potential and the actual membrane potential. This is because the series resistance is connected in series to the membrane resistance, and any applied voltage will distribute itself over these resistors proportional to their respective resistance values (Molleman, 2003). For example in a CHO cell voltage clamp experiment with a whole-cell current of 1nA and a series resistance of 10 MΩ a voltage discrepancy of 10 mV will be generated according to Ohms law ΔV = Rs x I. If the command potential is set to -60 mV, 10 mV will distribute over the series resistance so the actual membrane potential will be only -50 mV. In the presented CHO patch clamp recordings, series resistance was always < 10 MΩ and was compensated electronically by 80%.

In the Xenopus oocyte TEVC recordings currents were in the magnitude of several microamperes leading to the potential risk of a significant voltage drop occurring over the resistance of the ground electrode in the bath. An independent virtual-ground circuit was connected to the bath to clamp the bath potential to zero.

Series resistance also affects the speed of capacitative membrane charging/discharging upon a change in voltage. This can be expressed as:

Time constant of charging: τ = Rs x Cm

Since τ is directly proportional to the membrane capacitance, Cm and series resistance Rs of the cell, it follows that cells with large capacitance values and high series resistance levels are prone to significant delays in voltage changes, which in turn may lead to the masking of ion channel characteristics such as activation, inactivation and desensitization rates (Molleman, 2003). This was not of great concern in the presented electrophysiological experiments because no voltage-steps were implemented.

B. Junction potentials Junction potentials arise when dissimilar conductors are in contact. For example when lowering the pipette into the bath, differences in ionic concentrations and mobility between the pipette solution and the bath solution generate a liquid-liquid junction potential. In addition, redox reactions between metals and salt solutions can create a solid-liquid junction potential between the two media. This occurs at the liquid-metal junction between the pipette electrode and the pipette solution, and between the ground electrode and the bath solution (Molleman, 2003). These junction potentials can create artefacts in the form of an offset to the clamping potential and are therefore cancelled electronically by applying a potential of opposite polarity when the pipette is lowered into the bath solution. However, after obtainment of the whole-cell configuration the pipette is no longer in contact with the extracellular solution. This means that the actual liquid junction potential disappears while the applied compensation remains, creating an offset of equal size but opposite sign to the liquid junction potential (Figl, 2004). This was not corrected in the presented patch clamp

Concatenation of the Cys-loop receptors ______measurements. By use of the Clampex Junction Potential Calculator (pCLAMP 10.2, Axon CNS, Molecular Devices, Sunnyvale, CA, USA) the junction potential was estimated to -0.4 mV at 25 °C in the CHO cell patch clamp experiments and -0.2 mV at 20 °C for the Xenopus oocyte patch clamp experiments. These deviations were considered negligible in the presented data.

3.3.4 Patch clamp setup for CHO cell recordings Cell coated glass cover slips were transferred to a diamond shaped polycarbonate recording chamber (Warner Instruments, Hamden, CT, USA) of an inverted Olympus IMT-2 microscope placed on an anti-vibration table in a Faraday cage. The chamber was continually superfused with an extracellular standard Na-Ringer solution (appendix D2) and test solutions were applied through a custom made gravity driven Y-tube which was placed in the immediate vicinity of the cell of interest. The liquid-exchange time (the time span from the point when the test solution reaches the cell until the cell is totally bathed in test solution) for the Y-tube application system was < 100 ms. This was determined by a previous master student Marita G. Madsen via liquid junction potential measurements (Madsen, 2006). Pipettes were prepared from borosilicate capillary tubes (Sutter Instruments, Novato, CA, USA) and pulled by a DMZ-universal puller (Zeitz Instrumente, Bayern, Germany) to a final resistance of 1.5-3 MΩ. They were filled with intracellular standard K-Ringer solution (appendix D2) and mounted on the head stage of an EPC-9 amplifier (HEKA Instruments Inc., Bellmore, NY, USA) which was connected to a micromanipulator that enabled positioning of the pipette in the x-y-z plane (Eppendorf AG, Hamburg, Germany). The recording electrode inside the pipette was made of AgCl and was chlorided by electrolysis of the silver wire in a 0.9% NaCl bath. The reference electrode was an AgCl pellet electrode permanently placed in the recording chamber.

Whole-cell patch clamp recordings were performed at room temperature 16-40 hours after transient transfection of CHO cells. All recordings were performed at a holding potential of -60 mV. When currents exceeded 10 nA the holding potential was changed to less negative values to reduce the driving force for chloride. This occurred rarely and only for the GABAA ρ1 wildtype. The agonist pulse duration was ~2 seconds and the stimulation frequency was approximately one pulse per minute.

For the GABAA ρ1 wildtype receptor, recordings were performed before, parallel to and after the functional testing of concatamers to ensure that lack of concatamer current was not the result of an altered composition or concentration of the GABA solutions. Concentration-response measurements were made with GABA concentrations increasing from 0.1 to 316 µM in steps of ½ logC. Initial experiments were made with and without ASIC1a+3R to ensure that the co-transfection of the two receptors did not have any influence on the functional expression of ρ1 subunits. Furthermore it was confirmed that ASIC1a+3R was not activated upon application of GABA, and that GABAAR ρ1 was not activated upon application of acid.

Concatenation of the Cys-loop receptors ______

For the GABAA ρ1 dimers and trimers with different linkers a minimum of 5 transfections were stimulated with three concentrations of GABA: 1, 10 and 100 µM GABA. Each recording was repeated twice and when no current could be detected, Na-Ringer with pH 6.0 was applied to activate ASIC1A+3R and check the application system.

For the GABAA ρ1 L6ags concatamers a minimum of 8 cells from two transfections were stimulated with increasing concentrations of GABA ranging from 0.1 µM to 1 mM. Each recording was repeated twice and when no current could be detected, Na-Ringer with pH 6.0 was applied to activate ASIC1A+3R.

3.3.5 Patch clamp setup for Xenopus oocyte recordings 2-5 days post injection oocytes were transferred to flow cells which were continually superfused with standard oocyte Ringer’s solution (OR-2, appendix D2). Pipettes were prepared from fire-polished borosilicate tubes with filament (Sutter Instruments, cat# BF150-110-10) and pulled on a DMZ-universal puller (Zeitz Instrumente, Bayern, Germany) to a final resistance of 0.5-2 MΩ when filled with 1M KCl. Pipettes were mounted over Ag/AgCl-electrodes in pipette holders which were subsequently placed on headstages that could be manually controlled in the x-y-z plane via micromanipulators (World Precision Instruments, Sarasota, FL, USA). The headstages were connected to a GeneClamp 500 amplifier combined with a Digidata 1322A interface (both Molecular Devices, Sunnyvale, CA, USA). Drug-containing solutions were delivered to the flow cells via a custom-made syringe-driven system in which solutions were applied with a flow rate of 2.5 ml/min via a Gilson liquid handler 233 XL (Gilson Inc., Middleton, WI, USA). An Ag/AgCl pellet reference electrode was permanently placed in the bath.

Whole-cell patch clamp recordings were performed at 18-20°C at a holding potential of -60 mV. Generally, leak currents were negative and less than -200 nA. In rare cases where leak currents were greater, the holding potential was changed to -40 mV.

Maximal current amplitudes were determined by application of 1 mM GABA to a minimum of 8 oocytes per construct. All constructs were measured on the same day in oocytes from the same batch.

For GABA concentration-response measurements GABA was applied to the oocytes in increasing concentrations ranging from 1 nM-3.16 M with a lag period of approximately 4 minutes between stimulations to relieve the receptors of agonist-induced desensitization. The pulse duration was approximately 100 seconds. Measurements were made on a minimum of 6 oocytes per construct.

Diazepam concentration-response measurements was measured at a GABA concentration eliciting ~20% of the maximal current amplitude, and 3-4 GABA applications were performed before diazepam solutions were applied in increasing concentrations from 1 nM to 3.16 μM.

Concatenation of the Cys-loop receptors ______

3.3.6 Data analysis GraphPad Prism® 3.0 software (San Diego, CA, USA) was used to fit GABA or Diazepam concentration- response curves to the equation

n I/Imax = 1/(1+(EC50/[ligand]) )

where I stands for current, Imax is the maximal response, EC50 denotes the ligand concentration yielding 50% of the maximal response, and n is the Hill coefficient.

Diazepam potentiation was calculated by the equation

% Potentation = ((IGABA+DZ - IGABA) ∙ 100%)/IGABA

All results are presented as means ± standard error of the mean values.

For CHO patch clamp experiments data analysis and drawings were performed using the software IGOR (WaveMetrics, Lake Oswego, OR, USA) or GraphPad Prism® 3.0 (GraphPad Software, San Diego, CA, USA). For oocyte recordings pCLAMP software (Axon CNS, Molecular Devices, Sunnyvale, CA, USA) and GraphPad Prism® 3.0 software (San Diego, CA, USA) were used to analyze the data.

Statistical analysis was performed with One-way analysis of variance (ANOVA). Tukey’s post-test was applied for multiple comparisons of means. P values < 0.05 were considered significant and are denoted * in the figures. ** represents P < 0.01 and *** represents P < 0.001. Ns stands for “not significant” and n is the number of replicates.

______

4. RESULTS

The results are divided into two parts:

GABAA ρ1 concatamers: expression of homo-oligomeric GABAA concatamers in CHO cells. This section includes results from the linker optimization experiment, the molecular construction of the

GABAA ρ1 penta-concatamer and the assessment of functional expression of this construct.

GABAA α1β3γ2 concatamers: expression of hetero-oligomeric GABAA concatamers in Xenopus oocytes. This section will show results from determination of maximal current amplitudes and GABA- and diazepam concentration response experiments.

______

Concatenation of the Cys-loop receptors ______

4.1 GABAA ρ1 CONCATAMERS

4.1.1 GABAA ρ1 wildtype receptor

The homo-oligomeric GABAA ρ1 receptor was chosen as a model system to create a concatamer of five identical subunits. Transient transfection of CHO cells with cDNA encoding the untethered human ρ1 subunit + (80%), the H -activated ASIC1A+3 receptor (10%) and EGFP (10%) yielded robust inward current responses to GABA as assayed by whole-cell voltage clamp at -60 mV. This is depicted in figure 4.1A. The efficiency of transfection was ~75-90 % as estimated by the green fluorescence of cells when exposed to UV light.

A 0.01 μM GABA 0.1 μM GABA 0.316 μM GABA 1.0 μM GABA

-0.5 -0.5 -0.5

-1.0 -1.0 -1.0

nA nA nA

-1.5 3.16 μM GABA -1.5 10 μM GABA -1.5 31.6 μM GABA 100 μM GABA

-2.0 -2.0 -2.0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 s s s s

-0.5 -0.5 -0.5 -0.5

-1.0 -1.0 -1.0 -1.0

nA nA nA nA

-1.5 -1.5 -1.5 -1.5

-2.0 -2.0 -2.0 -2.0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 s s s s 1 nA B 3 s C

n = 14 1.0 20 n = 41 0.8 15 0.6 10 0.4

5 0.2 Number of cells

0.0 0 Peak current (% of GABAmax) (%of current Peak 0,01 0,1 1 10 100 1000 0-0.1 0.1-1 1-5 >5 M GABA Current (nA)

Fig. 4.1. Functional expression of GABAA ρ1 receptors in CHO cells. A: Representative current traces in response to 0.01 – 100 μM GABA. The bar above each trace indicates the approximate duration of one GABA application. B: GABA concentration-response curve. Means with SEM values from 4 separate transfections are shown. Individual curves were

normalized to the maximal current amplitude and averaged. C: Current distribution of GABAA ρ1 wildtype responses to 100 μM GABA. The cells were from more than five independent transfections.

Header and footer 1 15 December 2009 Concatenation of the Cys-loop receptors ______

The ρ1 wildtype receptors displayed a sigmoidal-shaped semi-logarithmic concentration response relationship with a GABA EC50 of 3.4 μM and a Hill coefficient of 2.2 ± 0.4 (fig. 4.1B). Previous heterologous

expression studies of human and rat ρ1 in HEK293 cells or Xenopus oocytes have reported GABA EC50 and Hill values in the ranges of 1-3 μM and 1.8-3.3, respectively (Amin and Weiss, 1996; Bormann, 2000; Enz and Cutting, 1999; Price et al., 2007; Rivera et al., 2000). The higher range GABA EC50 value found in this study might be due to a practical error in the patch clamp recordings. As can be seen in fig. 4.1A the duration

of agonist application did not outlast the slow activation of GABAA ρ1Rs at low agonist concentrations. Especially around the EC50 value it is evident that the agonist application was terminated before the peak response was reached. It is therefore quite likely that the peaks observed at 1 and 3.16 μM GABA would be higher at longer pulse durations, resulting in an agonist concentration response curve with a shallower slope and a lower GABA EC50 value. Variations in EC50 values and Hill slopes can also arise due to the use of different heterologous expression systems, or due to differences in the ligand application systems.

Responses were characterized by slow activation and deactivation and by a low degree of desensitization which increased at higher GABA concentrations. Maximal peak amplitudes evoked by 100 μM GABA were

predominantly in the nA-range as shown in fig. 4.1C, and GABAA ρ1R was functionally expressed in 100 % of the transfected cells. Therefore it was concluded that CHO cells mediate efficient heterologous expression of this receptor.

Exposure of the co-transfected ASIC1A+3R to pH 6.0 gave rise to large inward transients that were easily

distinguishable from GABAA ρ1 responses due to their fast activation, desensitization and deactivation kinetics. This was used as a positive control of the application system when no GABA-evoked currents could

be observed. Application of GABA in the 1-100 μM range did not activate ASIC1A+3R as illustrated in fig.

4.2., nor did application of Ringer with pH 6.0 activate GABAA ρ1R.

GABAA ρ1 R ASIC1A+3 R A B Ringer pH 6.0 100 μM GABA 100 μM GABA Ringer pH 6.0

0 0.0

-4 -0.5 -8 nA 4nA 2 nA -1.0 -12 nA 5 s 2,5 s

-16 -1.5

-20 Fig. 4.2. GABA and acid sensitivity of CHO cells transfec-2.0ted with GABAA ρ1R (A) or ASIC1A+3R (B). A: Transiently 0 10 20 30 40 transfected CHO cells expressing GABAs A ρ1Rs (90%) and EGFP (10%) displayed robust GABA-evoked current responses, but did not respond to application of Ringer with pH 6.0. B: Transiently transfected CHO cells expressing ASIC1A+3R (90%) and EGFP (10%) displayed robust acid-evoked current responses 0 but did not respond10 to application20 of 100 μM30 GABA. 40 s Measurements were made with whole-cell voltage-clamp. 57

Header and footer 1 03 January 2010 Concatenation of the Cys-loop receptors ______

4.1.2 Linker optimization The linker optimization study was performed to determine the optimal length of a linker for the succeeding construction of a GABAA ρ1 penta-concatamer. The hypothesis underlying this experiment was that a linker should ideally be short enough to prevent formation of dipentameric or hexameric receptors, but long enough to allow efficient assembly and functional expression of pentameric receptors on the cell surface. Titration of concatamers with different linkers could potentially reveal the optimal length by displaying a significant increase in functional expression upon co-transfection of dimer and trimer compared to the expression of each of these constructs separately. A similar approach was successfully applied by Baumann et al. (2001, 2002) for the concatenation of GABAA α1β2γ2 receptors.

Previously synthesized GABAA ρ1 concatenated dimers and trimers with different linker lengths were transiently transfected into CHO cells alone or in combination, and the functional expression of the constructs was tested via single-electrode whole-cell voltage clamp technology. The cells were voltage-clamped at -60 mV during all experiments and for each construct a minimum of 5 cells from at least two independent transfections were subjected to 1, 10 and 100 µM GABA.

*** 7000

6000

5000

4000 ns 3000

2000

1000

Maximal current amplitudes (pA) amplitudes current Maximal

(2) (3) (2) (3) (2) (3)

(2+3) (2+3) (2+3)

L30 (2) L30 (2) L40

L6ags (2) L6ags Monomer L20 L23 L25

GABAA 1 construct

Fig. 4.3. Maximal current amplitudes of GABAA ρ1 concatenated dimers and trimers in CHO cells. The average peak current of the constructs in response to 100 μM GABA was plotted. (2) denotes a dimer, (3) denotes a trimer, and (2+3) denotes co-expression of a dimer and a trimer in a 1:1 µg ratio. Lx denotes “linked by x amino acid residues”. L6ags denotes “linked by 6 repeats of AGS residues”. The means were plotted with error bars representing SEM values. ***: P < 0.001, ns: not significant.

Concatenation of the Cys-loop receptors ______

Fig. 4.3 shows the average of maximal peak amplitudes in response to 100 μM GABA for the GABAA ρ1 wildtype receptor and for each of the concatenated subunit combinations. GABA-evoked current responses between 20 pA and 300 pA were occasionally observed for all of the GABAA ρ1 concatamers except the L25 dimer and the L25 dimer + trimer. However, none of the linker constructs gave rise to mean current amplitudes in the wildtype magnitude (P < 0.001). One-way ANOVA with Tukey’s post test revealed no significant differences between the mean current levels of the 6 dimers tethered by different linkers (P > 0.05), nor was any significant difference observed between transfections with dimer, trimer or dimer + trimer with L20, L23 and L25 (P > 0.05). Thus, for these concatamer combinations the potential ability to form pentameric receptors did not improve the functional expression of constructs. Evidently, all the linkers seem to impair some vital step in the pathway from DNA transfection to functional surface expression of GABAA ρ1 receptors.

In a few cases, concatamers were able to form receptors of wildtype-like conductance with peak amplitudes between 500 pA and 2 nA. This is illustrated in fig. 4.4. The maximal current amplitudes of the GABAA ρ1 concatamers were divided into four categories of 0-100 pA, 100-500 pA, 500-1000 pA and >1000 pA to visualize the current distribution of the cell responses. From the graphs it can be deduced that the dimers linked with L30, L40 and L6ags gave rise to peak current amplitudes above 100 pA in the majority of cells in contrast to the other constructs. This result implies that L30, L40 and L6ags might allow functional expression of concatenated subunits to a greater extent than the other linker types. The fact that L6ags was 10-20 residues shorter than the two other functional linkers indicates that the difference in sequence design may provide this linker with a more favorable, flexible conformation. To address this hypothesis two linkers were chosen for the construction of the GABAA ρ1 penta-concatamer: L6ags and L30. The longest linker L40 was not chosen due to the risk of unconstrained subunit arrangement, and since the dimer + trimer combinations for L20, L23 and

L25 did not appear to increase the expression of functional constructs compared to the dimers or trimers alone, these linkers were not used for further experiments.

Concatenation of the Cys-loop receptors ______

11 11 11 10 L20 dimer 10 L20 trimer 10 L20 dimer + trimer 9 9 9 (n = 7) 8 (n = 8) 8 8 (n = 8) 7 7 7 6 6 6 5 5 5 4 4 4

3 3 3

Number Number of cells Number of cells 2 2 Number of cells 2 1 1 1 0 0 0 0-100 100-500 500-1000 >1000 0-100 100-500 500-1000 >1000 0-100 100-500 500-1000 >1000 Current (pA) Current (pA) Current (pA)

11 11 11 10 L23 dimer 10 L23 trimer 10 L23 dimer + trimer 9 9 (n = 9) 9 (n = 10) 8 (n = 6) 8 8 7 7 7 6 6 6 5 5 5 4 4 4

3 3 3

Number Number of cells Number of cells Number Number of cells 2 2 2 1 1 1 0 0 0 0-100 100-500 500-1000 >1000 0-100 100-500 500-1000 >1000 0-100 100-500 500-1000 >1000 Current (pA) Current (pA) Current (pA)

11 11 11 10 L25 dimer 10 L25 trimer 10 L25 dimer + trimer 9 (n = 5) 9 (n = 9) 9 (n = 7) 8 8 8 7 7 7 6 6 6 5 5 5 4 4 4

3 3 3

11 11 11 10 L30 dimer 10 L40 dimer 10 L6ags dimer 9 9 9 (n = 7) 8 8 (n = 15) 8 (n = 9) 7 7 7 6 6 6 5 5 5 4 4 4

3 3 3

Number Number of cells Number Number of cells Number Number of cells 2 2 2 1 1 1 0 0 0 0-100 100-500 500-1000 >1000 0-100 100-500 500-1000 >1000 0-100 100-500 500-1000 >1000 Current (pA) Current (pA) Current (pA)

Fig. 4.4. Current distribution of CHO cells transfected with L20, L23, L25, L30, L40 and L6ags concatamers. Maximal current levels in response to 100 μM GABA are shown for individual cells. The graphs are generated from the same data as fig. 4.3.

Concatenation of the Cys-loop receptors ______

4.1.3 Molecular construction of the GABAA ρ1 penta-concatamer with L6ags

A penta-concatamer of GABAA ρ1 subunits was made with the linker consisting of 6 Alanine-Glycine-Serine (AGS) repeats, each repeat encoded by the nucleotide sequence GCT-GGA-AGT. The linker region connecting two subunits in tandem was synthesized by overlap-extension PCR.

Figure 4.5A shows an example of primary overlap-extension PCR with ρ1C and ρ1E. ρ1C was incubated with

the tail primer L6agsas and the ρ1-specific end primer 935s (lane 1, expected size: 520 base pairs) or the ρ1- specific end primer 993s (lane 3, expected size 462 base pairs). Two different end primers were used to increase the chance of success. A negative control without template DNA was made for each reaction (lanes 2

+ 4). ρ1E was incubated with the tail primer L6agss and the ρ1-specific end primer 308as (lane 5, expected size:

300 base pairs) or the ρ1-specific end primer 327as (lane 7, expected size: 319 base pairs). Negative controls for these reactions are shown in lane 6 and 8.

Figure 4.5B shows a secondary PCR reaction with the templates [CC-L6ags] and [L6ags-EN]. The fragments were hybridized and elongated during the first 10 PCR cycles followed by addition of the end primers 993s and 327as for amplification of the 770 base pair fusion product (lane 1).

M 1 2 3 4 5 6 7 8 M M 1 2 A B Fig. 4.5. Synthesis of linker region A. Primary PCR reaction. 1: ρ1C with primer 935s + L6ags-as. 2: Negative control. 3: ρ1C with primer 993s + L6ags-as. 4: 850 Negative control. 5: ρ1E with primer 308as 650 + L6ags-s. 6: Negative control. 7: ρ1E with 500 primer 327as + L6ags-s. 8: Negative control. M: molecular size marker. B. Secondary 300 PCR reaction. 1: [CC-L6ags] + [L6ags -EN] with primers 993s + 327as. 2: Negative control. M: molecular size marker. C. For illustrative purpose two of the four linker templates generated during the first PCR reaction are marked in yellow and green. [C -L ]: 462 bps C C 6ags These were hybridized in the second PCR [L -E ]: 319 bps 5’ 3’ 6ags N reaction to create the linker region marked 3’ 5’ BsiWI 5’ 3’ in purple. ρ1 sequences are in dark blue, the 3’ 5’ AgeI unique digestion sites are in yellow and the 5’ 3’ 3’ 5’ synthetic linker is in red. [CC-L6ags-EN]: 770 bps

The concatenation of subunits was performed in two steps: first the subunits were ligated into the same vector and secondly the linker sequence was inserted between the subunits. Presence of the linker region in a

Concatenation of the Cys-loop receptors ______

concatamer was verified by digestion with XhoI and SacII. Correct insertion of this region deleted a XhoI site and introduced a SacII site between the coding sequences of two tandem subunits.

Figure 4.6 shows a XhoI digestion of the ligation intermediate ρ1 AB (lane 1) versus the linked dimer ρ1 L6ags A-B (lane 2). XhoI cleaves the AB construct in the pNS1zm vector region between the subunits and immediately downstream of the last subunit which in this case is B. This yields two fragments of 1461 and 5008 base pairs corresponding to the B subunit and the remaining plasmid, respectively. Insertion of the

linker region [AC-L6ags-BN] deletes the XhoI site located between the subunits, hence XhoI digestion of dimer

L6ags A-B only linearizes the construct.

1: A B M 1 2 1 2 M 1 21 : B M C D

7000 7000 5000 5000 2: A B 2: B C D

1650 1650 1000 Fig. 4.6. Insertion of linker 1000 Fig. 4.7. Insertion of linker region region deletes a XhoI site creates a SacII site between the

between the subunits. 1. subunits. 1. Digestion of L6ags BC- Digestion of AB with XhoI. D with SacII. 2. Digestion of trimer

2. Digestion of dimer L6ags L6ags B-C-D with SacII. M: A-B with XhoI. M: molecular size marker. molecular size marker.

Figure 4.7 shows a SacII digestion of ligation intermediate ρ1 BC-D versus the trimer ρ1 L6ags B-C-D. Construct BC-D contains two recognition sites for SacII; one located between the linked subunits C and D, and one in the 3’ end of D. Digestion with SacII yields two fragments: one of 1148 base pairs corresponding

to the region between the linker L6ags and the 3’ end of D (D2, see definition table 3.1), and another of 6752

base pairs corresponding to the remaining plasmid (lane 1). Insertion of the linker region [BC-L6ags-CN] to

yield the trimer L6ags B-C-D introduces a SacII site between subunits B and C. Therefore, digestion of the

trimer yields three fragments: one of 1431 base pairs corresponding to the region between the linkers in [BC-

L6ags-CN] and [CC-L6ags-DN], one of 1148 base pairs corresponding to the region between [CC-L6ags-DN] and D2, and finally one of 5294 base pairs corresponding to the remaining plasmid.

The presence and orientation of subunits was verified by multiple digestions with combinations of unique and vector-specific restriction enzymes as illustrated in fig. 4.8 and fig. 4.9. Fragments sizes were calculated from

the L6ags ρ1 concatamer plasmid maps and restriction site overviews in appendix B: Plasmids. The fragments excised from the vector are shown in green, yellow, purple, orange or turquoise and their sizes are given in the corresponding color below.

Concatenation of the Cys-loop receptors ______

Fig 4.8A shows three digestions of the dimer ρ1 L6ags A-B with BsrGI and SalI enzymes corresponding to A2 and B1 (lane 1), A1 and B2 (lane 2) and finally SacII and SphI which are linker- and vector-specific for this construct, respectively (lane 3). (See table 3.1 for definition of A1, A2, B1, B2 etc). Expected fragment sizes were: 1. 443 + 5999 base pairs, 2. 2469 + 3973 base pairs and 3. 72 + 575 + 1659 + 4136 base pairs.

Fragments < 100 base pairs were not visualized on the gels. The band patterns show that L6ags A-B was concatenated in the expected arrangement.

Fig. 4.8B shows the trimer ρ1 L6ags B-C-D digested with enzymes corresponding to B1 and C1 (lane 1), B2 and C1 (lane 2), C2 and D1 (lane 3) and SacII plus SphI for digestion of D2, linker and vector. Expected fragment sizes were: 1. 1497 + 6376 base pairs, 2. 524 + 7349 base pairs, 3. 437 + 7436 base pairs, 4. 72 + 511 + 575 + 1148 + 1431 + 4136 base pairs. The band patterns demonstrate the correct subunit alignment of the trimer ρ1 L6ags B-C-D.

Fig. 4.8C shows the tetramer ρ1 L6ags A-B-C-D digested with enzymes corresponding to A2 + B1 (lane 1), B2 + C1 (lane 2), A1 + B2 (lane 3), D2+ linker + vector (lane 4) and C2 + D1 (lane 5). Expected sizes of digestion products were: 1. 443 + 8864 base pairs, 2. 524 + 8783 base pairs, 3. 2469 + 6838 base pairs, 4. 72 + 511 + 575 + 1148 +1431 + 1434 + 4136 base pairs, 5. 437 + 8870 base pairs. Again, the band pattern was in congruence with the calculated fragment sizes.

Fig. 4.9A shows that all unique restriction sites were present in L6ags A-B-C-E-D. The pentamer was digested in five reactions with A2 + B1 (lane 1), B2 + C1 (lane 2), C2 + E1 (lane 3), E2 + D1 (lane 4), D2 + linker (lane 5), respectively. Expected sizes of fragments were: 1. 443 + 10301 base pairs, 2. 524 + 10220 base pairs, 3. 548 + 10196 base pairs, 4. 294 + 10450 base pairs, 5. 3 x 1434 + 1148 + 5294 base pairs.

Fig. 4.9B illustrates that the five subunits of the penta-concatamer were linked in the predicted alignment A- B-C-E-D. The construct was digested with A1 + B2 (lane 1), A1 + C2 (lane 2), A1 + E2 (lane 3) and A1 + D1 (lane 4). Expected sizes of fragments were: 1. 2469 + 8275 base pairs, 2. 3988 + 6756 base pairs, 3. 5568 + 5176 base pairs and 4. 5862 + 4882 base pairs.

In summary, five GABAA ρ1 subunits were successfully tethered on the DNA level by the L6ags linker. Each of the five ρ1 cDNAs contained two unique restriction sites, the presence of which was verified by multiple sets of restriction enzyme digestions. Fig. 4.10 illustrates this homo-oligomeric Cys-loop penta-concatamer in the pNS1zm vector. Positions of unique restriction sites, and sites used in the ligation process are shown.

Concatenation of the Cys-loop receptors ______

4.8A SacII

M 1 2 3 M

A1 A2 B1 B2 6000 3000 NheI BsrGI SalI PstI SphI SphI 1650

650 400 1: (443 bps) 300 2: (2469 bps) 3: (1659 + 575 bps)

4.8B SacII SacII

M 1 2 3 4 M B1 B2 C1 C2 D1 D2 SalI Pst I BspEI BsiWI BstBI SacII SphI SphI 2000 1650 1000 1: (1497 bps) 500 (524 bps) 400 2: 3: (437 bps) 4: (1431 + 1148 + 575 + 511 bps)

4.8C SacII SacII SacII

M 1 2 3 4 5 M A1 A2 B1 B2 C1 C2 D1 D2 8000 NheI BsrGI SalI PstI BspEI BsiWI BstBI SacII SphI SphI 3000 1650 1000 1: (443 bps) (524 bps) 500 2: 400 3: (2469 bps) 4:

(1431 + 1431+ 1148 + 575 + 511 bps) 5: (437 bps)

Fig. 4.8. Restriction enzyme verification of L6ags dimer, trimer and tetramer. A: L6ags A-B digested with 1: BsrGI + SalI, 2: NheI + PstI, 3:

SacII+SphI. M : molecular size marker. B: L6ags B-C-D digested with 1: SalI + BspEI, 2: PstI + BspEI, 3: BsiWI + BstBI, 4: SacII + SphI. M:

molecular size marker. C: L6ags tetramer A-B-C-D digested with 1: BsrGI + SalI, 2: PstI + BspEI, 3: NheI + PstI, 4: SacII + SphI, 5: BsiWI + BstBI. M: molecular size marker. The green, yellow, purple, orange and turquoise lines illustrate the lengths of the RE fragments generated from

each reaction, but do not show the size of the remaining vector. The ρ1 sequence is in dark blue with unique digestion sites in yellow and the linker in red. The arrows symbolize restriction enzymes.

Concatenation of the Cys-loop receptors ______

4.9A SacII SacII SacII SacII

A1 A2 B1 B2 C1 C2 E1 E2 D1 D2 NheI BsrGI SalI PstI BspEI BsiWI AgeI AclI BstBI SacII SphI SphI

1: (443 bps) (524 bps) 2: 3: (548 bps)

4: (294 bps)

5: (3x 1434 + 1148 bps)

M 1 2 3 4 5 M 1 2 3 4

10000 10000 7000 1650 5000 1000 4000 650 500 3000 300 A 2000 B

4.9B SacII SacII SacII SacII

A1 A2 B1 B2 C1 C2 E1 E2 D1 D2 NheI BsrGI SalI PstI BspEI BsiWI AgeI AclI BstBI SacII SphI SphI

1: (2469 bps) 2: (3988 bps) 3: (5568 bps) (5862 bps) 4:

Fig. 4.9. Restriction enzyme verification of L6ags pentamer. A. 1: BsrGI+SalI, 2: PstI + BspEI, 3: BsiWI + AgeI, 4: AclI + BstBI, 5: SacII. M: molecular size marker. B. Digestion with 1: NheI + PstI, 2: NheI + BsiWI, 3: NheI + AclI, 4: NheI + BstBI. M: molecular size marker. The coloured

lines illustrate the lengths of the restriction enzyme fragments generated from each reaction, but do not show the size of the remaining vector. The ρ1 sequence is in dark blue with unique digestion sites in yellow and the linker in red. The arrows symbolize restriction enzymes.

Concatenation of the Cys-loop receptors ______

4.1.4 Molecular construction of the GABAA ρ1 penta-concatamer with L30

The concatenation of GABAA ρ1 with L30 was performed as described for L6ags above. Linker regions were

successfully created by overlap extension PCR and ligation of the subunits ρ1A-E was initiated. However,

concatenation of the L30 pentamer is still in process, so no results will be presented for this construct.

A A

D2 B D B2

D1 C1 C E2 E C2

E1 5266

Fig. 4.10. Plasmid illustration of the GABAA ρ1 L6ags penta-concatamer A-B-C-E-D. Dark blue areas represent ρ1 subunits A- D, red areas represent L6ags and yellow areas represent unique restriction sites. The brown area represents the pNS1zm vector. Enzymes and positions of cleavage sites are shown. The unique restriction sites are underlined in yellow and denoted A1-2, B1-2, C1-2, E1-2 and D1-2 according to table 3.1.

Concatenation of the Cys-loop receptors ______

4.1.5 Electrophysiological analysis of GABAA ρ1 concatenated constructs

The GABAA ρ1 L6ags concatenated constructs were transiently transfected into CHO cells and functional expression was analyzed by serial application of GABA in the concentration range of 0.1 µM to 1 mM. A minimum of 8 cells from at least two transfections were tested with whole-cell voltage clamp at -60 mV.

As depicted in fig. 4.11 all L6ags concatamers showed impaired functional expression compared to the GABAA

ρ1 wildtype receptor. Virtually no current was seen for the trimer, trimer+dimer, tetramer or pentamer in response to 100 μM GABA. Statistically, there was no significant difference between the mean current amplitudes of any of the concatenated constructs (P > 0.05), and all the constructs displayed significantly lower maximal current amplitudes than the wildtype receptor (P < 0.001).

*** 7000 Fig. 4.11. Maximal current amplitudes of L 6000 6ags concatamers in CHO cells. The average peak 5000 current of the constructs in response to 100 μM GABA was plotted. Bars represent the mean of at 4000 least 8 measurements from two independent transfections, and error bars represent SEM (pA) 3000 ns values. ***: P < 0.001, ns: not significant. 2000

1000

0 Maximal current amplitudes

Monomer L6*ags dimer L6*ags trimer L6*ags tetramerL6*ags pentamer L6*ags dimer + trimer

Subunit combination

The current distribution of the 100 μM GABA responses from cells transfected with the L6ags constructs is shown in fig. 4.12. The current intervals are divided into 0-50 pA, 50-100 pA, 100 -500 pA and >500 pA. As was observed in the linker optimization experiment a substantial part of cells transfected with the dimer construct were responsive to GABA. For the L6ags concatamers the dimer produced both a higher frequency of positive responses and higher current levels than either of the other constructs. One might speculate that linkage of two subunits poses less constraints on receptor assembly than the linkage of three, four or five subunits, or that correct insertion of a transmembrane fusion protein into the plasma membrane becomes increasingly difficult the larger the protein.

Concatenation of the Cys-loop receptors ______

20 20 20 18 L6ags dimer* 18 L6ags trimer 18 L6ags dimer + trimer 16 16 16 14 n=18 14 n=10 14 n=8 12 12 12 10 10 10 8 8 8

6 6 6

Number of cells of Number cells of Number Number of cells of Number 4 4 4 2 2 2 0 0 0 0-50 50-100 100-500 >500 0-50 50-100 100-500 >500 0-50 50-100 100-500 >500 Current (pA) Current (pA) Current (pA)

L6ags pentamer n=24 20 L tetramer 20 18 6ags 18 16 16 14 n=9 14 12 12 10 10 8 8

6 6 Number of cells of Number 4 cells of Number 4 2 2 0 0 0-50 50-100 100-500 >500 0-50 50-100 100-500 >500 Current (pA) Current (pA)

Fig. 4.12. Current distribution of CHO cells transfected with GABAA ρ1 L6ags concatamers. Peak amplitudes in response to 100 μM GABA are shown for individual cells. The graphs are generated from the same data as fig. 4.11. Dimer: C-D, Trimer: B-

C-D, Tetramer: A-B-C-D, Pentamer: A-B-C-E-D. * This current distribution represents new L6ags dimer measurements (different than those presented in fig. 4.4).

For the penta-concatamer GABAA ρ1 L6ags A-B-C-E-D a few cells displayed GABA-evoked concentration- dependent whole-cell currents. As shown in fig. 4.13 the construct was indeed able to assemble into functional receptors with a GABA concentration-response relation similar to that of wildtype with respect to GABA EC50 and Hill coefficient. However, the current amplitudes were almost 100 fold lower than wildtype magnitude, and positive responses were found in only a minor part of the transfected cells, making further studies with this construct a difficult task. In attempt to improve expression, cells were transfected with DNA from two different midipreps and transfections were performed with the double amount of DNA with and without co-transfected ASIC1a+3R. Moreover, the incubation period of transfection was varied from 1-6 hours and patch clamp experiments were conducted 20-80 hours after transfection. The construct was also tested in transiently transfected HEK293 cells and in cRNA injected Xenopus oocytes. Unfortunately, none of these attempts proved fruitful. Due to time limitations of the project further attempts to increase the expression of the GABAA ρ1 L6ags A-B-C-E-D were postponed to future studies.

Concatenation of the Cys-loop receptors ______

A 0,1 μM GABA 1 μM GABA 3,16 μM GABA

0 0 0

-50 -50 -50

pA pA -100 -100 pA -100

-150 10 μM GABA -150 31,6 μM GABA -150 100 μM GABA

-200 -200 -200 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 s 0 s 0 s

-50 -50 -50

pA pA -100 pA -100 -100

-150 -150 -150

-200 -200 -200 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 s s s 100 pA

2s B 1.0 L6ags A-B-C-E-D

GABAA 1 WT 0.8 0.6 C GABAA ρ1 WT L6ags A-B-C-E-D 0.4 pEC50 5,465 5,514 0.2 EC50 3,424 μM 3,062 μM 0.0 Hill 2,229 2,831 Peak Current (% of GABAmax) (% of Current Peak 0,1 1 10 100 1000 M GABA

Fig. 4.13. Functional expression of GABAA ρ1 L6ags penta-concatamer A-B-C-E-D in CHO cells. A: Current traces in response to 0,1-100 μM GABA. B: Single-cell GABA concentration-response curve generated from the same data set as in A (red) and plotted

togetherHeader with and that footer of the GABAA ρ1 wildtype receptor (black, dotted). Baseline (9 pA)1 was subtracted from the GABA-evoked 08 December 2009 responses which were subsequently normalized to the maximal current amplitude. C: The pEC50 and Hill coefficients for the penta- concatamer and wildtype plotted in B. EC50 was calculated from pEC50. ______

4.2 GABAA α1β3γ2 CONCATAMERS

Hetero-oligomeric GABAA α1β3γ2 concatamers were functionally characterized in Xenopus oocytes by two- electrode voltage clamp. Previously synthesized dimers and trimers were expressed separately (β-α, γ-β-α), in combination (β-α + γ-β-α) or with free γ2 subunit (β-α + γ). cRNA was injected in concentrations that resulted in equal molar amounts of α and β subunits in the α/β mixture, the β-α mixture, the β-α + γ mixture and the α+β+γ mixture. Furthermore the molar amount of dimer (β-α) and trimer (γ-β-α) was the same for the constructs alone or in combination to enable comparison of the functional expression. When tethered in the trimer, γ was expressed in a 1:1 ratio to the α- and β-subunits resulting in the relative over-expression of γ due to the GABAA stoichiometry of two α subunits, two β subunits and one γ subunit per receptor. For the

Concatenation of the Cys-loop receptors ______wildtype α+β+γ receptor and the dimer + monomer combination (β-α + γ) the amount of γ was doubled in a 2:1 ratio relative to α and β, respectively. This was done to prevent the potential generation of a subpopulation of αβ receptors in the oocytes.

4.2.1 Maximal current amplitudes Maximal current amplitudes in response to 1 mM GABA were measured by two-electrode voltage clamp at -60 mV to assay the functional expression of the concatamers. All measurements were made on the same day on oocytes from the same batch injected two days previously. Statistical comparison was performed using One-way ANOVA with Tukey’s post test.

A B C *** ***

ns A) A) ns *** A) 20 20 20

15 15 15

10 10 10 ns

5 5 5

0 0 0

Maximal current amplitudes ( amplitudes current Maximal Maximal current amplitudes ( amplitudes current Maximal

- - 1:1 ( amplitudes current Maximal 1:1 1:1:2 - 1:1:2 1:1 1:1 - + - + + + + + + WT 1:1:2 + - - - + - Subunit combination + - Subunit combination Subunit combination

Fig. 4.14. Maximal current amplitudes of GABAA α1β3 and α1β3γ2 wildtype receptors and concatamers. 40 nl cRNA of each subunit combination was injected into Xenopus oocytes from the same batch approximately 40 hours before determination of the maximal current amplitude evoked by 1 mM GABA. Measurements were made with two-electrode whole-cell patch clamp technology at -60 mV. n = 8-14 for each construct. The ratios given for the wildtype, dimer + monomer and dimer + trimer combinations are in μg. A: Comparison of subunit combinations which were able or unable to form pentameric receptors. B: Comparison of receptors with or without γ2. C: Comparison of subunit combinations which were all theoretically able to generate pentameric receptors. The three graphs were generated from the same data set. ***: P < 0.001, ns: not significant.

Comparison of subunit combinations which were able or unable to form pentameric receptors As illustrated in fig. 4.14A the dimer + trimer combination, γ-β-α + β-α, produced a mean current amplitude of the same magnitude as the α+β+γ wildtype (P > 0.05). Thus, for the hetero-oligomeric GABAA concatameric constructs, linkage of the subunits did not cause the massive reduction in the functional expression that was seen for the homo-oligomeric ρ1 concatamers. Expression of the dimer or trimer alone on the other hand resulted in dramatic decreases in the functional expression (P < 0.001), indicating that

Concatenation of the Cys-loop receptors ______formation of a pentameric α1β3γ2 receptor was more favourable with respect to expression than formation of tetramers or hexamers with dangling subunits.

Properties of the γ subunit From figure 4.14A it can furthermore be deduced that oocytes injected with the trimer (γ-β-α) cRNA displayed higher current levels than those injected with the dimer (β-α) although the difference was not significant. Increased expression of γ2 containing receptors was also observed for receptors composed of free

α1, β3 and γ2 subunits as depicted in fig. 4.14B compared to receptors composed of only α1 and β3. This demonstrates the enhancing role of γ2 for trafficking of receptors to the membrane surface (Essrich et al., 1998).

Comparison of pentameric subunit combinations

Figure 4.14C shows three subunit combinations that theoretically give rise to pentameric α1β3γ2 receptors. It is evident that the α1+β3+γ2 wildtype combination and the dimer + trimer combination displayed significantly higher current levels than the dimer + monomer combination (P < 0.001). This is unexpected since the molar ratio of γ2 to α1 and β3 is the same for the dimer + monomer combination and the α1+β3+γ2 wildtype (a 1:1 β-α

+ γ ratio in μg corresponds to a molar α:β:γ ratio of 1:1:2). Apparently, the γ2 subunit did not co-assemble as efficiently with the dimers as with the untethered subunits. Furthermore, the formation of functional receptors composed of dimers + trimers was more successful than the formation of receptors composed of dimers + monomers. The relatively low degree of functional expression of the β-α + γ combination could potentially be due to degradation of the in vitro synthesized cRNA for this subunit combination.

4.2.2 GABA concentration-response measurements In order to evaluate whether concatenation had any effect on the potency of GABA for the surface-expressed receptors, GABAA α1β3γ2 concatamers were expressed in Xenopus oocytes and exposed to increasing concentrations of GABA ranging from 0.1 μM to 3.16 mM. GABA-evoked peak amplitudes were measured using two-electrode whole-cell voltage clamp technique at -60 mV and for each oocyte the responses were normalized to the maximal current amplitude observed. Representative traces are shown in fig. 4.15. The data was plotted in Graph Pad Prism to create sigmoidal concentration-response curves with variable Hill slope as shown in fig. 4.16. The pEC50 of individual oocytes was determined and values for each construct were subsequently averaged. EC50 was calculated from the mean by the formula: EC50 = 10-pEC50. The results are plotted in table 4.1. Statistical comparison of pEC50 values by One-way ANOVA with Tukey’s post test is depicted in fig. 4.16A-C.

Concatenation of the Cys-loop receptors ______

GABAA α+β WT 1:1 GABAA α+β+γ WT 1:1:2 0.1 0.3 1.0 3.16 10 31.6 100 316 0.1 0.3 1.0 3.16 10 31.6 100 316 1000

2 μA 2 μA

4 min 4 min

GABAA β -α GABAA β–α + γ 1:1 0.3 1.0 3.16 10 31.6 100 316 0.3 1.0 3.16 10 31.6 100 316 1000 3160

4.316730.624856.076810.00000 min min min min -7-1407 nA nA2-6 nA nA

2 μA 2 μA

4 min 4 min

GABAA γ -β –α GABAA γ -β –α + β –α 1:1

0.1 0.3 1.0 3.16 10 31.6 100 316 1000 3160 0.3 1.0 3.16 10 31.6 100 316 1000

1 μA 2 μA

4 min 4 min

Fig. 4.15. GABA-evoked current responses of GABAA α1β3γ2 concatamers expressed in Xenopus oocytes. The representative traces for each recording were clustered. In real time the applications were separated by 4 minutes to relieve the receptors of agonist-evoked desensitization. The bars above each trace indicate the approximate duration of application.

Header and footer 1 03 February 2010 Fig. 4.15 shows that all subunit combinations gave rise to GABA-evoked concentration-dependent macrocurrents in the μA range. Responses were characterized by fast activation and by increasing desensitization with increasing GABA concentration. For example the dimer + trimer receptors (γ-β-α + β-α) generated a non-desensitizing current in response to 1 μM GABA, whereas GABA concentrations above 3.16 μM evoked desensitizing responses for this construct. It is evident that the dimer and trimer concatamers were

able to assemble into functional GABAA receptors with agonist-evoked response kinetics resembling those of wildtype receptors, even when expressed separately.

Concatenation of the Cys-loop receptors ______

A 1.0 1+ 3 WT 1:1 1+ 3+ 2 WT 1:1:2 A 0.8 3- 1 + 2 1:1 0.6 2- 3- 1 3- 1 + 2- 3- 1 1:1 0.4 3- 1 0.2

0.0 Peak current (% max) current of GABA Peak 0.01 0.1 1 10 100 100010.000 M GABA

6.5 6.5 *** B ** C 6.0 6.0 **

5.5 *** 5.5 pEC50 5.0 pEC50 5.0

4.5 4.5

4.0 4.0

1:1 + + 1:1:2 1:1:2 + + + + +

Subunit combination Subunit combination

Fig. 4.16. Potency of GABA on GABAA α1β3γ2 concatamers and wildtype receptors. A: GABA concentration-response curves of GABAA α1β3γ2 wildtypes and concatamers expressed in Xenopus oocytes, fitted with variable Hill slope. Mean values for 6-14 oocytes from4.2.3 one Diazepam injected batch concentration are shown for each-response subunit combination. Individual curves were normalized to the maximal current amplitude and averaged. Error bars represent SEM values. B-C: Statistical comparison of pEC50 values for GABAA α1β3γ2 concatamers and wildtype receptors. Bars represent means and error bars represent SEM values. **: P < 0.01, ***: P < 0.001, ns: not significant. B:

Properties of the γ subunit, C: Comparison of pentameric subunit combinations.

Properties of the γ subunit Fig. 4.16B shows a significant decrease in pEC50 for the γ-containing receptors. This was both the case for receptors composed of free subunits (P < 0.01) and for pseudo-hexameric receptors composed of concatenated trimers (P < 0.001). Evidently, the γ subunit has a negative influence on the potency of GABA on these receptors. A similar observation was made by Boileau et al. (2003). Furthermore, dimers and trimers

Concatenation of the Cys-loop receptors ______expressed separately resulted in the expression of receptors with significantly lower GABA sensitivity than either of the wildtype combinations (P < 0.001). This indicates that receptors composed of an even number of subunits are unable to bind or gate as efficiently as pentameric receptors.

Comparison of pentameric subunit combinations From fig. 4.16C it can be deduced that linkage of subunits decreased the pEC50 values of expressed receptors even when a pentameric arrangement was possible. This is also illustrated by the rightward shift of GABA concentration-response curves for the concatenated constructs relative to the two wildtype receptors in fig. 4. 16A. Particularly the dimer + monomer combination displayed very low GABA pEC50 values compared to the α+β+γ wildtype. It can be speculated whether this is due to a low degree of γ incorporation, resulting in expression of receptors made from dimers predominantly. However, as can be seen by comparing fig. 4.16B and C even receptors composed of dimers solely displayed a higher GABA pEC50 than the dimer + monomer combination.

Hill coefficients for the GABA concentration response curves were > 1 for all subunit combinations indicating that all the receptors should be able to cooperatively bind two GABA molecules. This is shown in table 4.1. The coefficients were however quite variable. The α1+β3+γ2 wildtype receptors and the receptors composed of dimer + trimer were characterized by steeper slopes of ~ 2.3 while the α1+β3 wildtype receptors had a slope of 1.7. Receptors composed of the dimer or the trimer both had slopes of 1.4. Baumann et al. (2002) observed lower Hill coefficients of 1.4 and 1.04 for the αβγ wildtype receptors and for the dimer + trimer receptors, respectively. Furthermore the GABA EC50 values reported for these two subunit combinations were 10 and 25 times higher than the corresponding values reported in this study, respectively.

This discrepancy could be due to different properties of the two β subunit isoforms (β2 in their study versus β3 in this study), or more likely, due to differences between the application systems and the experimental setup.

4.2.3 Diazepam concentration-response measurements

As mentioned previously the binding site for benzodiazepines on GABAA receptors resides at the interface between the + side of an α subunit and the – side of a γ subunit. This means that benzodiazepines only modulate GABA-evoked currents of receptors that incorporate the γ subunit on the + side of an α subunit. The benzodiazepines do not by themselves induce responses, but increase the frequency of channel opening induced by GABA (Sigel, 2002). Potentiation therefore depends on the correct formation of GABA binding sites between the + face of a β subunit and the – face of an α subunit. Modulation of the GABAA α1β3γ2 constructs by diazepam was evaluated to assess the correct arrangement and stoichiometry of the concatenated receptors.

Concatenation of the Cys-loop receptors ______

Concentration-response measurements were generated by applying increasing concentrations of diazepam ranging from 1 nM to 3.16 µM to the oocytes in the presence of a GABA concentration corresponding to EC20 as estimated from the GABA concentration-response curves in fig. 4.16. For each oocyte 4 initial GABA EC20 applications were delivered without diazepam, and % potentiation was calculated by the formula: ((IGABA+DZ-IGABA) x 100 %)/IGABA. Data was plotted in Graph Pad Prism and the potentiation values were averaged for each construct. Curves in fig. 4.17A depict the mean of normalized responses for each construct. Maximal potentiation values (Emax) are shown in table 4.1. Statistical comparison of these values is depicted in fig. 4.17B.

+ + WT 1:1:2 300 1 3 2 3- 1 + 2 1:1 2- 3- 1 200 3- 1 + 2- 3- 1 1:1

3- 1

100 Potentiation (%) Potentiation

0 0.001 0.01 0.1 1 10 M Diazepam

ns Fig. 4.17. Diazepam modulation of GABAA * α1β3γ2 wildtype and concatamer receptors. B 400 A: Diazepam concentration-response curves. Means with SEM values for 5-9 oocytes from 300 one batch are shown for each subunit combination. Individual curves were 200 normalized to the current in the absence of diazepam and subsequently averaged. B: Statistical analysis of diazepam maximal

% % Potentiation 100 potentiation values (Emax) for GABAA α1β3γ2 0 concatamers. Means of Emax are plotted with - error bars representing SEM values. The - 1:1:2 - graphs were generated from the same data set + - + - as A. *: P < 0.05, ns: not significant. -

Subunit combination

From fig. 4.17A it can be concluded that diazepam was able to potentiate GABA-evoked currents for all the

γ2-containing subunit combinations. As expected, receptors composed of the β3-α1 dimer were not sensitive to

75 Header and footer 1 14 February 2010 Concatenation of the Cys-loop receptors ______

diazepam. For the dimer + trimer γ-β-α + β-α, the trimer γ-β-α and the α+β+γ wildtype a maximal diazepam potentiation of 200-350% was observed. As depicted in fig. 4.17B no statistical difference between these three constructs could be found although the dimer + trimer seemed to be the most diazepam-sensitive combination. The findings correlate well with results obtained by Baumann et al. (2002) and demonstrate that tethering of the γ subunit ensures its correct incorporation into the receptors. GABA-evoked current responses of cells expressing the dimer + monomer combination (β-α + γ) were subjected to significantly less diazepam mediated potentiation than currents of any other γ-containing construct, indicating that the γ subunit was not correctly incorporated into the majority of these receptors.

Large variations in the degree of diazepam potentiation were observed for each construct, as illustrated by the S.E.M. values of % potentiation on the diazepam concentration-response curves. This could be due to the formation of subpopulations of receptors without the γ subunit, or due to variations in the potency of GABA on individual oocytes injected with the same cRNA construct. Application of a GABA concentration outside the steep part of the GABA concentration-response curve for a given oocyte generates a response near plateau or baseline, which reduces the diazepam modulation window. Baumann et al. (2002) performed diazepam concentration response measurements at a GABA concentration evoking 2-5% of the maximal current amplitude. In contrast, the corresponding measurements of this study were made at a GABA concentration evoking ~20% of the maximal current amplitude.

GABA + GABA DIAZEPAM

Subunit combination Emax ± SEM n EC50 pEC50 ± SEM Hill ± SEM n Emax ± SEM n

µA µM %

α1+β3 WT 1:1 4.53 ± 0.99 14 1.18 5.93 ± 0.07 1.65 ± 0.09 14 - -

α1+β3+γ2 WT 1:1:2 14.09 ± 1.68 8 2.64 5.58 ± 0.07 2.29 ± 0.23 13 183.33 ± 68.11 8

β3-α1 0.56 ± 0.19 11 11.35 4.95 ± 0.11 1.42 ± 0.31 6 - -

β3-α1 + γ2 1:1 5.75 ± 0.85 12 30.27 4.52 ± 0.06 1.65 ± 0.12 11 78.70 ± 8.87 7

γ2-β3-α1 3.59 ± 0.76 11 61.17 4.21 ± 0.03 1.42 ± 0.07 10 206.31 ± 24.80 6

γ2-β3-α1 + β3-α1 1:1 15.49 ± 1.46 11 7.33 5.14 ± 0.11 2.26 ± 0.16 13 283.60 ± 53.54 8

Table 4.1. Properties of GABA and diazepam on GABAA α1β3γ2 concatenated dual and triple constructs expressed in Xenopus oocytes. Measurements of maximal GABA-evoked current amplitudes were performed on the same day with oocytes from a single batch. The GABA and diazepam concentration response measurements were made on oocytes from only one batch for each subunit combination. The GABA EC50 value was calculated from the mean pEC50. All measurements were made with two-electrode voltage-clamp.

Concatenation of the Cys-loop receptors ______5. DISCUSSION

The discussion is divided in two sections corresponding to 4: Results.

GABAA ρ1 concatamers expressed in CHO cells. This section includes the linker optimization

experiment and the penta-concatenation of GABAAR ρ1.

GABA α1β3γ2 concatamers expressed in Xenopus oocytes. ______

5.1 GABAA ρ1 CONCATAMERS

5.1.1 Linker optimization In several cases it has been shown that successful concatenation is dependent on the properties of linkers used to tether the subunits (Baumann et al., 2001; Carbone et al., 2009; Zhou et al., 2003). An optimal linker should be short enough to constrain the stoichiometry and arrangement of subunits while long enough to enable efficient assembly, trafficking and surface expression of receptors. Furthermore, the linker should be unable to interfere with the ligand binding sites of the receptor or to disrupt the conformational changes associated with gating. In order to identify such a linker for the penta-concatenation of GABAA ρ1 a linker optimization experiment was performed at the onset of this project. 6 different linkers of 20 to 40 amino acid residues tethering GABAA ρ1 subunits in dimers and trimers were functionally screened by whole-cell voltage clamp measurements in CHO cells. Unfortunately all the linkers impaired the functional expression of

GABAA ρ1 receptors. Specifically, co-expression of L20, L23 and L25 dimers and trimers did not increase the GABA-evoked current levels compared to the separate expression of these constructs, as would be expected upon the correct formation of a pentameric receptor complex. This outcome could be due to a suboptimal length or sequence of the linkers.

A. Comparison of linker lengths

In a GABAA α1β2γ2 concatenation study by Baumann et al. (2001) it was implied that the length of a functional linker depends on the magnitude of the N- and C-terminal protrusions of subunits from the plasma membrane. By experimental titration of linker lengths analogously to present optimization experiment they found an “actual linker length” of 23-27 amino acids to be optimal for the tethering of GABAA α1 and β2 subunits. This length included the C-terminal protrusion of the first subunit in a tandem and accounted for variations in the lengths of the N-terminal protrusion of α1 and β2 subunits prior to the conserved α helix seen

“on top” of the extracellular domain in fig. 1.7. In another concatenation study of α4β2 nAChRs by Zhou et al. (2003) it was shown by sucrose gradient sedimentation combined with electrophysiological measurements

Concatenation of the Cys-loop receptors ______that dimers linked by 32 amino acid residues or more were able to form functional dipentamers when expressed separately. Co-expression with free subunits resulted in the exclusive formation of monopentamers. In this case, the linker length included the C-terminal protrusion, introduced restriction sites and the synthetic peptide, but did not account for differences in the lengths of the extracellular domains of nAChR α4 and β2 subunits. In order to enable comparison of these linker experiments with the one conducted in present study, the entire extracellular N- and C-terminal protrusions of linked subunits together with lengths of the synthetic peptides are given in table 5.1. The data in this table was provided by The Universal Protein Resource online database in December 2009 (UniprotKB, 2009). “C’ prot.” denotes the predicted amino acid stretch spanning from the last residue of transmembrane segment M4 to the end of the first subunit in a tandem, whereas “N’ prot.” denotes the predicted length of the N-terminal domain preceeding the first amino acid of transmembrane segment M1, excluding the signal sequence of the second subunit in a tandem. This is illustrated in fig. 5.1. It is important to note here that these lengths are hypothetical and based on the amino acid sequences of subunits rather than on crystallographic studies. Hence the exact number of residues contributing to each region may deviate.

As can be seen in table 5.1 the linkers used in the concatenation of GABAAR α1β2γ2 by Baumann et al. (2001, 2002) produced a total length from M4 of the first subunit in a tandem to M1 of the second subunit in a tandem of 242-257 amino acid residues. Similarly, in the concatenation study by Zhou et al. (2003) the distance bridging M4 of nAChR α4 with M1 of β2 or vice versa was 240 or 258 residues long. In both groups small currents were observed upon expression of dimers alone, however co-expression with free subunits resulted in a substantial increase in functional expression. Thus it appears as if dimers with a total M4→M1 length of approximately 240-260 amino acid residues may be able to form functional dipentamers on the cell surface, but mono-pentamers are assembled by a more efficient mechanism upon co-expression of the dimers with free subunits.

From table 5.1 it is also evident that GABAA ρ1 subunits seem to have a substantially larger N-terminal domain than the other subunit types. All ρ1 concatamers were connected by a total length of 280 to 300 amino acid residues from M4 to M1, which means that theoretically each of the synthetic linkers introduced in these constructs should be long enough to enable pairwise assembly of dimers and trimers into functional pentameric receptors. One reason why the functional expression of GABAA ρ1 concatamers in CHO cells was severely reduced compared to the wildtype receptor could be that the 6 linkers chosen for the GABAA ρ1 concatamers were actually too long, complicating the efficient folding or assembly of the subunits in the E.R.

Concatenation

Concatenation of the Cys-loop receptors ______

Fig. 5.1. Illustration of the terms used for linker the comparison of total linker lengths in table 5.1. Two Cys-loop subunits, each with a large extracellular domain, 4 N’ prot. transmembrane segments and a short C- terminal tail, are linked from C to N by a C’ prot. synthetic linker (red, dotted). The pore- lining M2 segment is in dark red. C’ prot. denotes C-terminal protrusion, N’ prot. M1 M2 M3 M4 M1 M2 M3 M4 denotes N-terminal protrusion and corresponds to the extracellular domain excluding the signal peptide. Figure modified from (Jensen et al.., 2003).

Study Receptor Construct C' prot. Linker N’ prot.* Total length Baumann et al., 2001+2002 α-10-β 13 10 219 242 GABAAR

α1β2γ2 (rat) α-10-γ 13 10 234 257 β-23-α 1 23 223 247 γ-26-β 1 26 219 246

1 Z hou et al., 2003 α -6ags-β 8 24 208 240 nAChR α4β2 (human) β-6ags-α 24 20 214 258

This study ρ1-20-ρ1 0 20 260 280 Header and footer 4 GABAAR ρ1 11 February 2010 (human) ρ1-23-ρ1 0 23 260 283 ρ1-25-ρ1 0 25 260 285 ρ1-30-ρ1 0 30 260 290 ρ1-40-ρ1 0 40 260 300 ρ1-6ags-ρ1 0 20 260 280

Table 5.1. Comparison of linker optimization experiments. *Predicted length of extracellular domain - signal peptide. The

numbers denote the number of amino acid residues predicted to contribute to a given subunit region. Concatamers are named by

the first subunit in a tandem followed by the linker length in amino acid residues, and the second subunit of the tandem. “6ags” stands for “6 repeats of alanine-glycine-serine”. The total length is the sum of the C-terminal protrusion (C’ prot.), the synthetic linker and the N-terminal protrusion (N’ prot.) and denotes the predicted length from the end of the M4 transmembrane segment of the first subunit in a tandem to the beginning of the M1 transmembrane segment of the second subunit in the tandem. The data was extracted in December 2009 from the online protein sequence database: The Universal Protein Resource (UniprotKB, 2009).

B. Potential formation of loop-over structures Based on the crystallized structure of AChBP (Brejc et al., 2001) and the cryo-EM images of the Torpedo nAChRs (Unwin, 2005) it can be estimated that the N-terminus of Cys-loop subunits is located at the “top” of the extracellular domain approximately 62 Å away from the C-terminal protrusion, which is predicted to be located near the membrane. This illustrated in fig. 5.2. A linker connecting two subunits from C to N has to

Concatenation of the Cys-loop receptors ______

span at least the corresponding distance. Secondary structures of the linker and/or short C- and N-terminal protrusions of the tandem subunits would increase the required linker length, as would also be the case if the N- and C-termini to be linked project away from each other at the subunit interface. Thus a minimal linker length of approximately 70-80 Å is probably more realistic.

Baumann et al. (2001) calculated the “actual length” of their functional GABAA α-10-β linker to be ~83 Å with 3.6 Å per amino acid residue in an extended conformation. This “actual length” included the C-terminal protrusion and the synthetic linker as shown in table 5.2. When calculating the “actual linker length” of their

β-23-α construct they additionally included the N-terminal elongation of α1 relative to that of β2 (4 amino acid

residues) to take into consideration the variation in length of the extracellular domains of α1 and β2 when comparing α-β and β-α concatamers. By this approach an “actual linker length” corresponding to ~100 Å was obtained for the β-23-α construct (table 5.2). To evaluate whether these linker lengths were excessive enough to enable interspersing of a free subunit between the two subunits of a tandem (fig. 5.2), they furthermore estimated the minimal length of a polypeptide passing the perimeter of a Cys-loop subunit. Based on the cryo- EM images of Torpedo nAChRs this length was assumed to be > 54 Å (Miyazawa et al., 1999). Together these estimates led to the conclusion that the linkers used in the α-10-β and β-23-α constructs were probably short enough to keep the concatenated subunits of a tandem in adjacent positions in the pentamer.

GABAA tandem α-10-β β-23-α ρ-(20-40)-ρ Predicted C-terminal protrusion of the first subunit 13 a.a. 1 a.a. 0 a.a. Synthetic linker 10 a.a. 23 a.a. 20-40 a.a.

N-terminal elongation relative to β2 0 a.a. 4 a.a. 41 a.a. Actual length with 3.6 Å per a.a. 83 Å 100 Å 220-292 Å

Table 5.2. Calculation of ”actual linker length” according to Baumann et al.. (2001). The “actual linker length” is the sum of the amino acid residues that constitute the C-terminal protrusion, the synthetic linker and the N-terminal elongation relative to that of the

β2 subunit. An extended conformation of 3.6 Å per amino acid residue is assumed. a.a.: amino acid residues. Å: Ångström.

Using the same calculation approach for the GABAA ρ1 concatamers and taking into account that the ρ1

subunit has a N-terminal elongation of 41 amino acid residues relative to the GABAA β2 subunit, the “actual linkers” used in present optimization study obtain lengths of 220- 292 Å (table 5.2). This is probably long enough to allow interspersing between or “looping over” of linked subunits, especially if the small N-terminal α helix (fig. 1.7) is flexible. In such a rearranged scenario the linkers could easily disrupt the interactions between the N-terminal interfaces of subunits during receptor assembly. A similar hypothesis was proposed

by Ericksen and Boileau (2007). They used a molecular mechanical model of the GABAA tandems from Baumann et al. (2001) to show that the looping-over of a tethered subunit in a pentameric arrangement does not necessarily pose as much strain on the linker as expected. It was speculated that the use of excessive

Concatenation of the Cys-loop receptors ______

linker lengths could promote the formation of misassembled structures, thereby reducing the functional expression of concatamers. Should this be the case in present study, it can be argued that the shortest linker,

L20, should be more functional than the longer, L25, which was not observed. However, due to the large

extracellular domain of the GABAA ρ1 subunit even L20 may be excessive in length. With this in mind it could

be interesting to tether GABAA ρ1 subunits directly from C to N without a synthetic linker.

Fig. 5.2. Unconstrained subunit arrangement due to an excessive linker A B length. Three neighbouring subunits of a ~ 62Å pentamer are shown in side view (A) and from the top (B). The green subunit is > 54 Å interspersed between the linked blue 1 subunits. The M1-M2, M2-M3 and M3- 4 3 M4 loops are omitted for clarity. The 3 linker is in dotted red. The pore-lining M2 1 3 1 4 segment is in dark red. From the cryo EM M1 M4 M4 M3 1 3 4 M4 images of Torpedo nAChRs the height of the extracellular domain is estimated to 62 Å and according to Baumann et al.. (2001) the perimeter of a subunit is > 54 Å.

C. Potential lack of C-terminal protrusion It is a prerequisite for successful concatenation that the N- and C-terminal of the subunits to be linked are located on the same side of the plasma membrane. If the final amino acid residue of the C-terminus of

GABAA ρ1 resides within the plasma membrane as indicated in table 5.1, the presence of a linker could Header and footer seriously interfere with the correct1 folding and membrane insertion of16 Februarythe second 2010 and/or third subunit of a dimer or trimer concatamer. This would impair the expression of constructs irrespective of the linker length

used. Substituting the final amino acid residue of the GABAA ρ1 C-terminus with a cysteine residue and probing the covalent modification of this residue with a water-soluble sulfhydryl-specific agent could establish whether it protrudes on the extracellular side of the plasma membrane.

D. Potential degradation of linker It can be hypothesized that some unexpected property of the linker sequence results in E.R. retention of the

GABAA ρ1 subunits or in internal cleavage of the fusion polypeptide, producing non-functional, truncated subunits. However, the amino acid composition of the 6 linkers used in the optimization experiment was based on sequences which were successfully applied by Baumann et al. (2001, 2002) and by Zhou et al. (2003). Alanine, glutamine and proline residues were interspersed between two repeats of five glutamines to

create the uncharged, flexible linkers L20, L23, L25, L30 and L40 whereas 6 repeats of the sequence alanine–

glycine-serine were used to create L6ags with a random coil structure. Pure polyglutamine linkers were avoided to prevent exhaustion of the glutamine tRNA pool. Moreover, signal peptides of all but the N-terminal subunit

Concatenation of the Cys-loop receptors ______in a concatamer were deleted and stop codons in the linker stretch were removed to prevent premature termination of translation. The possibility of intracellular degradation due to the presence of the linker cannot be completely excluded since Western blots were not performed in this study to document the integrity of concatenated constructs. This was attempted, but the GABAA ρ1 antibody used showed a very high degree of unspecific binding. The occasional generation of GABA-evoked current by the ρ1 constructs which was independent of concatamer stoichiometry could be due to functional assembly of minor breakdown products. However, no linker proteolysis has been detected by Western blots in other studies of Cys-loop concatamers as long as the signal sequences of all but the first linked subunit were omitted (Minier and Sigel, 2004b).

5.1.2 GABAA ρ1 penta-concatenation

A. Low functional expression of higher-order concatamers

As for L23, L25 and L30 concatenation with L6ags severely impaired the functional expression of GABAA ρ1 in

CHO cells even when a pentameric arrangement was possible. A few cells transfected with the L6ags penta- concatamer did produce GABA-evoked currents, in one case with a pEC50 and Hill value close to that of wildtype ρ1 receptors. However, since this was a rare event it was not possible to physiologically or pharmacologically characterize this construct, nor was it plausible to use it for further experiments such as determination of the number of agonist molecules required to fully activate homo-oligomeric Cys-loop receptors.

Reduced functional expression of penta-concatenated constructs has been reported in several studies of hetero-oligomeric Cys-loop receptors. For example nAChRs concatenated in the arrangement β4-β4-α3-β4-α3 by Sivilotti and group displayed only 50% of wildtype current levels in oocytes although the amount of injected cRNA was 100-fold greater than that of the wildtype subunits (Groot-Kormelink et al., 2006). In a

GABAAR concatenation study where α1-β2-α1-γ2-β2 cRNA was injected in comparable amounts to free α1, β2 and γ2 subunits in oocytes concatenation reduced the functional expression of receptors to 40% of the wildtype level (Baur et al., 2006). Upon transient transfection of the same construct into HEK293 cells the maximal current amplitude was further reduced to only 1-2% of the wildtype amplitude. For comparison, expression of the corresponding dimer + trimer combination in oocytes yielded maximal current amplitudes of wildtype magnitude whereas transient transfection into HEK293 cells resulted in peak currents that were approximately 30% of the wildtype level (Baur et al., 2006). Kaur et al. (2009) also observed a 30% reduction in functional expression in Xenopus oocytes upon concatenating GABAA β-α-δ/β-α trimers + dimers into the pentameric arrangement β-α-δ-β-α. Together these results point to a negative correlation between the number of linked subunits and the functional expression of constructs. Moreover, they imply that mammalian cells are

Concatenation of the Cys-loop receptors ______less suitable for the functional expression of concatamers compared to oocytes. Potential explanations for these two indications are discussed below.

B. Potential disruption of the assembly pathway As mentioned in section 1.1.6A it is believed that oligomerization of Cys-loop receptors involves the stepwise formation of distinct assembly intermediates. Linkage of subunits might interfere with such a process resulting in E.R. retention and reduced expression of receptors. The heterodimer model proposed for nAChRs suggests that assembly begins with the formation of two dimers followed by association of the final subunit to create a pentamer (Blount et al., 1990; Gu et al., 1991; Saedi et al., 1991). Should this be a general scenario for the assembly of Cys-loop receptors it is plausible that the co-expression of dimers and trimers is more successful than expression of penta-concatamers. It can be hypothesized that concatenation of only two or three subunits enables minor rotations and dynamic interactions between the linked subunits to expose motifs important for successful receptor assembly and bury motifs responsible for E.R. retention. Perhaps such subunit flexibility is hindered by the increased constraint caused by linkage of multiple subunits in a penta- concatamer. The sequential nAChR assembly model suggests the formation of trimers in a rapid, possibly co- translational manner, followed by the sequential association of the two remaining subunits to create a pentamer (Green, 1999). Penta-concatenation may also interfere with such a process, since the linked subunits are unable to associate co-translationally. If the N-terminal subunit of a penta-concatamer polypeptide associates with the N-terminal subunit of another penta-concatamer polypeptide before the following subunits on each string are translated an aggregate would most likely form in the E.R. Therefore higher-order concatenation involves the potential risk of wrong assembly, leading to E.R. retention. Incorporation of a fluorescent tag in the concatamer constructs and visualization of their subcellular localization by confocal laser scanning microscopy could reveal whether E.R. retention is the cause of low functional penta- concatamer expression. In vitro translation of the constructs combined with blue native PAGE could furthermore reveal the oligomeric state of the translation products.

C. Choice of expression system

The CHO cell line was chosen for the expression of GABAA ρ1 concatamers since the wildtype receptors gave rise to large GABA-evoked macro-currents in 100% of transiently transfected CHO cells. However, none of the GABAA ρ1 concatenated constructs displayed currents of wildtype magnitude in CHO cells. In contrast,

GABAA α1β3γ2 concatamers were efficiently expressed in Xenopus oocytes and displayed maximal current amplitudes in the μA range. Notably the functional expression of the dimer + trimer combination (β-α+ γ-β-α) was as high as that for the α+β+γ receptors composed of untethered subunits. This supports the hypothesis that oocytes may provide a more efficient heterologous expression system for concatamers than mammalian

Concatenation of the Cys-loop receptors ______cells. Assaying the functional expression of GABAA α1β3γ2 concatamers in CHO cells could be interesting in this aspect.

The CHO cell line has not been applied for concatamer expression in previous Cys-loop receptor studies. Xenopus oocytes have on the other hand been a popular choice as shown in table 1.3. The large size of these cells enables easy electrophysiological manipulation and direct injection of in vitro synthesized cRNA into their cytoplasm. This overrules potential problems with the uptake of large plasmid constructs that might occur in mammalian cell transfections and excludes the risk of incomplete cDNA transcription. Besides, the low ambient temperature (18-20°C) of oocytes could be beneficial for the folding of large concatamers. It has been shown that the assembly of Torpedo nAChRs is badly impaired at 37°C due to misfolding of the subunits, whereas lowering of the temperature to 26°C facilitates expression (Paulson and Claudio, 1990). The same principle could apply for concatamers. Alternatively, a different repertoire of chaperone proteins in oocytes may promote surface expression of some concatenated constructs. Unfortunately, injection of the

GABAA ρ1 penta-concatamer cRNA into oocytes failed to improve its functional expression as did transient transfection into HEK293 cells, implying that the lack of current was due to dysfunction of the construct itself rather than some incompatibility of the CHO cell line with respect to concatamer expression.

5.1.3 In summary

Overall the data presented in this thesis indicates that the GABAA ρ1 receptor is not suitable for concatenation. The functional expression of this receptor was badly impaired upon concatenation and could not be improved by changing the linker length or sequence, the number of linker subunits or the expression system. Since no biochemical validation experiments were performed it was not possible to determine whether this inefficiency was due to faulty translation of the construct or due to posttranslational aggregation and retention in the E.R as a result of inefficient folding, assembly or glycosylation. Intracellular proteolysis, impaired trafficking or dysfunctional agonist binding and gating of surface-expressed receptors could also explain the lack of current. Either way, identification of the faulty step is important for successive changes in concatenation design that might improve the functional expression of this construct.

5.2 GABAA α1β3γ2 CONCATAMERS

5.2.1 Functional expression Successful functional expression of concatamers has in many cases been used as an indication of correct receptor stoichiometry and arrangement. Exemplifying this are the two studies conducted by Baumann et al.

Concatenation of the Cys-loop receptors ______in 2001 and 2002, which are described in section 1.2.2.A of the introduction. However, successful functional expression might not always reveal the “true” architecture of receptors.

In present study dimer and trimer concatamers of GABAA α1, β3 and γ2 subunits produced GABA-evoked current amplitudes in the μA range when expressed separately. This indicates that these constructs were able to assemble into functional GABA sensitive receptors on the cell surface although formation of a pentameric arrangement should theoretically not be possible. As mentioned previously, dimers may assemble in two pairs bridged by a fifth dimer to create dipentamers on the cell surface as shown in fig.5.3G. Such dipentamers would contain receptors of two stoichiometries: one of (α)3(β)2 and one of (α)2(β)3. According to Baumann et al. (2001) only the latter is functional. However, co-expression of GABAA α-β tandems with free α subunits has proven successful in concatenation studies by Im et al. (1995) and Boileau et al. (2005), implying that a

GABAAR stoichiometry of (α)3(β)2 is also functional. Alternatively, the separate expression of β-α dimers or γ-β-α trimers could generate monopentamers with a “dangling” subunit sticking out of the receptor complex as shown in fig. 5.3 or the constructs could be proteolyzed to generate assembly-competent breakdown products.

In congruence with our findings, the Sigel group recently reported GABA-evoked current amplitudes of 0.5-2

μA upon separate expression of β3-α1 dimers and γ2-β3-α1 trimers in Xenopus oocytes (Baur et al., 2009; Kaur et al., 2009). Intriguingly, they did not observe such artefactual currents upon expression of the same amount of these constructs with the β3 subunit replaced by β2 (Sigel et al., 2009). Thus, it seems that the β3 subunit may specifically promote the surface-expression of GABAA pseudo-hexamers or dipentamers.

The potency of GABA on β-α and γ-β-α constructs was significantly decreased compared to their respective wildtype counterparts (α+β and α+β+γ). This indicates that the affinity and/or efficacy of the agonist were reduced for dipentameric or pseudo-hexameric assembly formations, or the gating mechanism of these receptors could be altered. The trimer construct was however able to assemble into diazepam-sensitive receptors containing at least one intact β-/+α GABA binding site and one α+/-γ benzodiazepine binding site (Baur and Sigel, 2005).

5.2.2 Incomplete incorporation of subunits The fact that dimer and trimer were able to assemble into functional receptors alone raises the question: what receptor types are responsible for the GABA-evoked current produced when these constructs were combined with each other or with a free γ subunit? Co-expression of the dimer and the trimer significantly increased the maximal current amplitudes to α+β+γ 1:1:2 wildtype level as seen in Baumann et al. (2002). Formation of a pentameric arrangement is clearly more favourable than the formation of dipentamers or hexamers by each of these constructs alone. The arrangement is shown in fig. 5.3D. However, co-expression of dimers with free γ

Concatenation of the Cys-loop receptors ______

subunit did not increase the functional expression of receptors to the same extent, although the maximum current amplitude of β-α + γ was higher than that of β-α alone. Apparently, assembly of the dimers with the untethered γ subunit was less efficient than assembly with the trimer, resulting in a subpopulation of receptors composed of dimers only. This was also reflected by a low degree of diazepam potentiation of this subunit combination compared to that of the dimer + trimer.

A B Fig. 5.3. Theoretical GABAA α1β3γ2 subunit combinations arising from monomers, dimers and trimers.

A: α+β+γ wildtype receptor composed GABAA GABAA of two α (dark blue), two β (light blue) α+β+γ α+β

2 and one γ subunit (grey). B: α+β

β 2 wildtype receptor composed of two α and three β subunits. For both illustrations, the GABA binding sites are denoted by blue arrows. The red arrow shows the benzodiazepine binding site which is confined to γ- C D containing receptors. The (+) and (-) γ α_ γ α _ interface of each GABA binding site is + β β + in red. C: Correct assembly of two β-α + β β - + dimers with one γ subunit. D: Correct α - α assembly of one γ-β-α trimer with one β-α + γ γ-β-α + β-α β-α dimer. Both arrangements produce receptors with two GABA binding sites and one benzodiazepine binding site. E E F γ α_ α β α_ & F: Suspected assembly of dimers or + trimers alone to form pseudo-hexamers + β β β β + with dangling subunits. Both GABA + - - α α γ binding sites are intact. Only F is sensitive to benzodiazepines. G: when β-α γ-β-α expressed separately, five dimers can assemble to form functional G dipentamers. For C-G the synthetic +β _ α α β α_ linker is shown in dotted red.

_ + β+ α β β + - α β-α

Header and footer 1 04 February 2010 Variable incorporation of the untethered γ subunit was additionally suspected in the oocytes injected with free α+β+γ subunits in a 1:1:2 ratio. The GABA pEC50 value of this subunit combination was intermediate to that of γ-β-α + β-α and α+β and the maximum diazepam potentiation of α+β+γ receptors was lower than that of the dimer + trimer combination, implying the presence of α+β receptors within the α+β+γ population. A

similar scenario was observed by Czajkowski and group. Specifically, they found that coinjection of GABAA

Concatenation of the Cys-loop receptors ______

α1, β2 and γ2 subunits in a 1:1:1 ratio into Xenopus oocytes resulted in reduced, variable and diminishing diazepam sensitivity over time compared to oocytes injected with α1+β2+γ2 in a 1:1:10 ratio, and the GABA

EC50 for the 1:1:1 combination was in between that of pure α1β2 receptors and that of α1β2γ2 1:1:10 (Boileau et al., 2003).

In present study, the γ2 subunit was expressed in a 1:1 μg ratio to the β-α dimer. This ratio is generally applied in our lab when using these concatamers. Since it takes two dimers and one γ subunit to create a diazepam- sensitive GABAA receptor as shown in fig. 5.3C this resulted in a molar α:β:γ ratio of 1:1:2. Sigel and group expressed the same dimer + monomer combination in an α:β:γ ratio of 1:1:10. They observed a maximum current amplitude comparable to that of α+β+γ 1:1:5 wildtype receptors and a maximum diazepam potentiation almost 100% higher than that of wildtype receptors (Baumann et al., 2001, 2002). Thus it appears that the γ subunit must be over-expressed in a 10 fold relation to α and β to ensure its incorporation, if it is not tethered.

5.2.3 Reorientation of dimers Intriguingly, the pEC50 value of the dimer + monomer combination was not only significantly lower than that of the α+β+γ wildtype and the dimer + trimer combination, it was also lower than that of the β-α dimer. Should the β-α + γ combination give rise to a mixed population of receptors composed of either the dimer alone or the dimer combined with γ, it would be expected that the pEC50 value would lie in between that of the dimer and that of the dimer + trimer. The fact that it did not suggests that a part of the receptors expressed from this subunit combination were less functional than receptors composed of dimers alone. Baumann et al. (2002) also observed a rightward shift in the concentration-response curve of β-α + γ relative to α+β+γ, although the magnitude of this shift was not as great as observed in present study. It is assumed that two dimers combine with a γ subunit to form the counterclockwise arrangement γ-β-α-β-α when viewed from the synaptic cleft as shown in fig. 5.3C. Alternatively, dimers assemble in dipentamers or pseudohexamers as shown in 5.3E+G. All these arrangements give rise to receptors with two functional GABA binding sites, each located at the interface between the (+) side of a β subunit and the (-) side of an α subunit in a dimer construct.

Should the two subunits within a dimer be able to switch positions so that a β+--α construct turns into –α-β+ the agonist binding site within the dimer is lost (fig. 5.4). This is possible if the linker and the amino acid termini of the subunits are flexible enough to allow rotation of the alpha subunit “around” the beta subunit. Assembly of such rearranged tandems with a γ subunit could result in receptors of the counterclockwise arrangement: γ--

α-β+--α-β+ as shown in fig. 5.4. In this assembly version only one agonist binding site remains intact. Since it has been shown that activation of receptors with one binding site is about 60 times less efficient than activation of wildtype receptors, the presence of receptors in such a rearrangement could potentially explain the low GABA pEC50 and the low maximum current amplitude observed for the β-α + γ combination.

Concatenation of the Cys-loop receptors ______

B Fig. 5.4. Dimer rearrangement. γ +β The subunits* of a dimer may switch positions. β - α The+ color code is similar to fig. 5.3. The - α numbers 1.1-4 indicate the four transmembrane

1 segments of each subunit. For simplicity,

4 2

3 - M1-M4 are only shown for one dimer of the γ α receptor- complex. The (+) and (-) face of the αGABAβ + binding site are located within the β + 2. * dimer (in red). If the two subunits switch - places as indicated by the black arrows, the * GABA binding site within the dimer is lost.

Thisγ γ rearrangement is denoted by *. The 1

βrearrangement+ γ results in receptors with only 2 4 - α

3 one functional GABA binding site located 3. - between the dimers. *

Re-orientation of dimer constructs was also suspected by the Sigel group who observed that α+--β constructs were able to form diazepam sensitive receptors upon co-expression with γ although the α+/-γ binding site for benzodiazepines was expected to be lost upon concatenation (Baumann et al., 2001). A similar observation was made by Boileau et al. (2005) and by Im et al. (1995). However, no biphasic GABA or diazepam

concentrationHeader and footer -response curves were3 obtained for the 01 β January-α 2010 + γ combination in present study as would be expected if a subpopulation of receptors with only one agonist binding site was present. Furthermore, the Hill coefficient of the β-α + γ GABA concentration-response curve indicated the presence of more than one intact binding site on the receptors. Thus, the existence of surface-expressed, rearranged receptors within the β-α + γ population is only speculative.

5.2.4 In summary Obviously, successful functional expression is not the only criterium that must be fulfilled in order to establish a useful concatenation system. It is important that the concatenated receptors contain intact ligand binding sites and display current kinetics that are similar to wildtype receptors, if they are to be used as model systems. For example a significantly altered sensitivity towards agonist or modulators might be the result of a distorted receptor architecture, which makes it difficult to interpret on the effects of introduced mutations.

In this study, the oocytes injected with the dimer + trimer combination behaved much like those injected with α+β+γ subunits in a 1:1:2 ratio. The functional expression and maximal diazepam potentiation of the γ-β-α + β-α receptors was slightly increased whereas the potency of GABA on these receptors was decreased. Since the γ subunit decreases the GABA pEC50 of receptors as observed in fig. 4.16B (Boileau et al., 2003) and

Concatenation of the Cys-loop receptors ______participates in the binding of benzodiazepines, this indicates that γ was incorporated in all receptors of the dimer + trimer type in contrast to the untethered receptors. Similar findings were reported by Baumann et al. (2002). The dimer + trimer combination thus provides an example of a successful concatenation system. However, the observation that dimers and trimers are by themselves able to generate GABA-evoked currents is a bit concerning. It cannot be completely excluded that these constructs form dipentamers or pseudo- hexamers within a γ-β-α + β-α population, although it is probably less likely. Furthermore, co-expression of β-α with γ does not ensure a stoichiometry of γ-β-α-β-α as expected, since the dimers appear to assemble as efficiently with each other as with the free subunit when co-expressed in a 1:1 ratio. Additionally, the risk of re-orientation of the two subunits within a dimer questions the validity of such a construct. Clearly, a penta- concatamer provides the most predictable and reliable arrangement of the five subunits and is the optimal goal in order to obtain Cys-loop receptors of predefined stoichiometry. Therefore, future studies within this field should be dedicated to optimize the functional expression of such constructs.

Concatenation of the Cys-loop receptors ______6. CONCLUSION

GABAA ρ1 concatamers The linker optimization experiment did not reveal any significant difference between the six linkers.

L6ags and L30 which were 20 and 30 amino acid residues long, respectively, were chosen for the

GABAA ρ1 penta-concatenation.

A homo-oligomeric GABAA ρ1 penta-concatamer was successfully constructed with the L6ags linker.

The penta-concatamer linked by L30 is still in process.

Functional expression of the ρ1 penta-concatamer linked with L6ags was extremely low in CHO-K1 cells and could not be improved by transient transfection of the construct into HEK293 cells or by cRNA injection into Xenopus oocytes. Therefore it was not possible to use this construct to determine the number of agonist molecules required to fully activate a homo-oligomeric Cys-loop receptor.

GABAA α1β3γ2 concatamers

The GABAA β-α dimer and γ-β-α trimer were able to generate GABA-evoked current upon separate expression in Xenopus oocytes, presumably by assembling into dipentamers or pseudo-hexamers. The functional expression and GABA sensitivity of these constructs was significantly lower than those of the corresponding wildtype receptors. The maximum diazepam potentiation of the trimer was similar

to that of the α1β3γ2 1:1:2 wildtype receptor.

Co-expression of the β-α dimer and the γ-β-α trimer produced receptors that were as efficiently

expressed in Xenopus oocytes as was the α1β3γ2 GABAA wildtype receptor. The potency of GABA on the β-α + γ-β-α combination was lower than that of α+β+γ 1:1:2 whereas the maximum diazepam potentiation was higher, presumably due to a variable incorporation of the γ subunit into the untethered wildtype receptors.

The functional expression and sensitivity towards GABA and diazepam was significantly reduced for the β-α + γ combination compared to the wildtype receptors. Apparently, the γ subunit must be tethered or over-expressed in a 10 fold relation to α and β to ensure its incorporation into receptors.

Overall, it is concluded that the GABAA γ-β-α + β-α dimer + trimer combination provides a reliable

means of expressing GABAA α1β3γ2 receptors of defined stoichiometry and arrangement. Co- expression of the β-α dimer with free γ subunit in a 1:1 μg ratio does however not produce a homogenous population of receptors of the γβαβα arrangement, as was previously expected.

Concatenation of the Cys-loop receptors ______7. FUTURE DIRECTIONS

As mentioned in the discussion, the low functional expression of GABAA ρ1 concatamers calls for a series of biochemical experiments to establish the reason for this. These include Western blots to check for premature termination of translation and intracellular degradation, SCAM to determine the location of the final residue of the ρ1 C-terminus, scanning confocal microscopy to check for E.R. retention, and in vitro translation combined with blue native PAGE or sucrose gradient sedimentation to check the oligomerization status of the constructs. Binding assays could additionally reveal potential surface-expression of non-functional receptors, and concatenation of GABAA ρ1 with a shorter linker could eliminate the possibility of non-functional subunit “loop-over” arrangements.

The GABAA α1β3γ2 concatamer experiments in Xenopus oocytes should be repeated to obtain results from two separate batches of oocytes, and perhaps the functional expression of constructs should be normalized to that of an endogenous ion channel to account for variations in expression levels between individual oocytes as was done by the Sigel group (Baumann et al., 2001, 2002). Furthermore it would be interesting to elucidate differences between β2 and β3 subunits that might explain the isoform-dependent generation of artefactual dimer and trimer currents reported by Sigel et al. (2009), and to express GABAA α1β3γ2 concatamers in CHO cells to examine the general ability of this cell line to express concatamers. Coinjecting the γ subunit in a 10:1 relation to free α and β subunits or to β-α dimers is also desirable to establish whether this does indeed ensure incorporation of the γ subunit as proposed.

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101

Concatenation of the Cys-loop receptors ______APPENDIX A: MEMBRANE POTENTIAL & CURRENT

Neuronal signalling involves rapid changes in the electrical potential across the plasma membranes of neurons. At rest, this potential exists due to concentration gradients of ions across the semi-permeable plasma membrane which are maintained by the concerted action of resting channels and pumps such as the leak K+ channels and the Na+/K+ ATPase (Ashcroft, 2000). Intracellular and extracellular ion concentrations of a typical mammalian cell are shown in mM below.

Ion Extracellular concentration mM Intracellular concentration mM Equilibrium potential mV Na+ 135-145 12 +66 K+ 3.5-5 140 -93 Ca2+ 2.25-2.52 10-7 M (free) +135 Cl- 115 2.5-50 -42

Extracellular ion concentrations refer to the range found in human blood. Intracellular ion concentrations refer to those of a typical mammalian cell. The equilibrium potentials are calculated for 37°C and the middle of the concentration range indicated. The table is from Ashcroft (2000).

The equilibrium potential for a single ion, X, denotes the potential at which no net transmembrane flux of X occurs, and depends on the concentration gradient and valence of X. The equilibrium potential for X is defined by the Nernst Equation:

(Hille, 2001) where R is the gas constant (8.3145 J K-1 mol-1), T is the absolute temperature, z is the valence of X, F is -1 Faraday’s constant (96485 C mol ), and [X]out and [X]in are the extracellular and intracellular concentrations of ion X, respectively.

Normally, the resting membrane is permeable to more than one ionic species, predominantly Na+, K+ and Cl-.

In order to account for this, the resting membrane potential (Erest) can be described by the Goldman-Hodgin- Katz (GHK) equation:

(Hille, 2001) where R is the gas constant (8.3145 J K-1 mol-1), T is the absolute temperature, F is Faraday’s constant (96485 -1 C mol ), P is the permeability coefficient of a given ion and [X]out and [X]in are the extracellular and intracellular concentrations of ion X, respectively.

102

Concatenation of the Cys-loop receptors ______

Generally, the resting membrane potential of a mammalian cell is in the range of -50 mV to -80 mV (Molleman, 2003).

Upon appropriate chemical or electric stimulation, ligand- or voltage-gated ion channels in the plasma membrane will open transiently and allow fast diffusion of selected ions through their pores. This will drive the membrane potential in the direction of the equilibrium potential(s) (Ex) for the permeant ion(s) (Molleman, 2003). When the membrane potential equals the equilibrium potential of a permeant ion X, the electrical driving force exactly balances the chemical driving force acting upon X, hence the net flow of X across the plasma membrane will be zero. For ion channels such as GABAARs which are almost exclusively permeable to a single ion species (X = Cl-), this state is experimentally defined as the reversal potential. For the less selective cation channels such as the nAChRs the reversal potential is a function of both the relative permeability and the equilibrium potentials of the permeant ions (X = Na+, K+) (Molleman, 2003).

The magnitude of ion flux is determined by the conductance (g) and the electrochemical driving force (E) acting upon the permeant ion X. The conductance is defined as the reciprocal of resistance (R): whereas the driving force is defined as the difference between the membrane potential (Emembrane) and the equilibrium potential for X: .

Ohm’s law states that voltage (E) is the product of current (I) and resistance (R):

(Hille, 2001)

This rule can be modified to describe the current-voltage relationship of ion channels:

(Hille, 2001)

The macroscopic current that is recorded in whole-cell patch clamp experiments reflects the current passing through multiple channels in the plasma membrane. The size of this current depends on the number of channels in the membrane (n), the open state probability of these channels (P0) and the unitary current (i):

(Hille, 2001)

Ideally, the number of channels in the membrane (n) remains constant during patch clamp recordings. The unitary current (i) can however vary depending on the driving force acting upon the permeant ion. In voltage clamp experiments the driving force is kept constant, hence changes in the macroscopic current are assumed to reflect changes in the open state probability (P0). The open state probability in turn reflects gating of the channels.

103

Concatenation of the Cys-loop receptors ______APPENDIX B: PLASMIDS

B1: L6ags dimer A-B in pNS1zm

Enzyme Frequency Position BsrGI 1 2045 EcoRI 3 942 (747),1689 (1434),3123 NheI 2 885 (107),992 PstI 1 3461 SacII 1 2398 SalI 1 2488 SpeI 1 925 XbaI 1 3820 XhoI 1 3814

104

Concatenation of the Cys-loop receptors ______

B2: L6ags trimer B-C-D in pNS1zm

Enzyme Frequency Position BsiWI 1 3546 BspEI 1 2551 BstBI 1 3983 EcoRI 4 942 (747),1689 (1434),3123 (1431),4554 NheI 1 885 PstI 1 2027 SacII 3 2398 (1431), 3829 (1148), 4977 SalI 1 1054 SpeI 1 925 XbaI 1 5251 XhoI 1 5245

105

Concatenation of the Cys-loop receptors ______

B3: L6ags tetramer A-B-C-D in pNS1zm

Enzyme Frequency Position BsiWI 1 4980 BspEI 1 3985 BsrGI 1 2045 BstBI 1 5417 EcoRI 5 942 (747),1689 (1434),3123 (1434),4557 (1431),5988 NheI 2 885 (107), 992 PstI 1 3461 SacII 4 2398 (1434),3832 (1431),5263 (1148),6411 SalI 1 2488 SpeI 1 925 XbaI 1 6685 XhoI 1 6679

106

Concatenation of the Cys-loop receptors ______

B4: (L6ags tetramer A-B-C-E in pNS1zm)

Enzyme Frequency Position AclI 1 6560 AgeI 1 5528 BsiWI 1 4980 BspEI 1 3985 BsrGI 1 2045 EcoRI 5 942 (747), 1689 (1434), 3123 (1434), 4557 (1434), 5991 NheI 2 885 (107), 992 PstI 1 3461 SacII 3 2398 (1434), 3832 (1434), 5266 SalI 1 2488 SpeI 1 925 XbaI 1 6688 XhoI 1 6682

107

Concatenation of the Cys-loop receptors ______

B5: L6ags pentamer A-B-C-E-D in pNS1zm

Enzyme Frequency Position AclI 1 6560 (10744) AgeI 1 5528 (10744) BsiWI 1 4980 (10744) BspEI 1 3985 (10744) BsrGI 1 2045 (10744) BstBI 1 6854 (10744) EcoRI 6 942 (747),1689 (1434),3123 (1434),4557 (1434),5991 (1434),7425 (4261) NheI 2 885 (107),992 (10637) PstI 1 3461 (10744) SacII 5 2398 (1434),3832 (1434),5266 (1434),6700 (1148),7848 (5294) SalI 1 2488 (10744) SpeI 1 925 (10744) SphI 3 8359 (575),8934 (72),9006 (10097) XbaI 1 8122 (10744) XhoI 1 8116 (10744)

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Concatenation of the Cys-loop receptors ______

B6: The pNS1z vector CMV: Cytomegalovirus promoter ColE1: E.coli Origin of replication EM7: Bacterial promoter pA: polyA signal SV40: Simian virus promoter T7: promoter MCS: Multiple cloning sites f1: Filamentous bacteriophage Origin of replication ZEO: zeocin resistance gene

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Concatenation of the Cys-loop receptors ______APPENDIX C: PROTOCOLS

C1. Macherey-Nagel NucleoSpin® Extract II Protocol for direct purification of PCR products

1. Adjust DNA binding conditions Mix 1 volume of sample with 2 volumes of buffer NT (e.g. mix 100 µl PCR reaction with 200 µl NT). For sample volumes < 100 µl adjust the volume of the reaction mix to 100 µl using buffer NT or water. For removal of DNA fragments > 65 bp, dilutions of buffer NT can be used instead of 100 % NT.

2. Bind DNA Place a NucleoSpin® Extract II Column into a 2 ml NucleoSpin Collecting Tube and load the sample. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin® Extract II Column back into the collecting tube.

3. Wash silica membrane Place a NucleoSpin® Extract II Column into a 2 ml NucleoSpin Collecting Tube and load the sample. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin® Extract II Column back into the collecting tube.

4. Dry silica membrane Centrifuge for 2 min at 11,000 x g to remove buffer NT3 quantitatively. Make sure the spin column doesn’t come in contact with the flow- through while removing it from the centrifuge and the collecting tube. Residual ethanol from buffer NT3 might inhibit subsequent reactions and has to be removed in this step. In addition to centrifugation, total removal can be achieved by incubation of NucleoSpin® Extract II Columns for 2-5 min at 70°C prior to elution.

5. Elute DNA Place the NucleoSpin® Extract II Column into a clean 1.5 ml microcentrifuge tube. Add 15-50 µl elution buffer NE and incubate at room temperature for 1 min to increase the yield of eluted DNA. Centrifuge for 1 min at 11,000 x g. Yield of larger fragments (>5-10 kb) can be increased by using prewarmed elution buffer (70°C): For elution, add prewarmed elution buffer and incubate at room temperature for 1 min before collecting eluate by centrifugation.

C2. Macherey-Nagel NucleoSpin® Extract II Protocol for DNA extraction from agarose gels

1. Excise DNA fragment Take a clean scalpel to excise the DNA fragment from an agarose gel. Excise gel slice containing the fragment carefully to minimize the gel volume. Determine the weight of the gel slice and transfer it to a clean tube.

2. Gel lysis For each 100 mg of agarose gel add 200 µl buffer NT. For gels containing >2 % agarose, double the volume of buffer NT. The mazimum amount of gel slice per NucleoSpin® Extract II Column is 400 mg or 200 mg of a high percentage gel >2%. In this case 2 loading steps are required 8step 3). Incubate sample at 50 °C until the gel slices are dissolved (5-10 min). Vortex the sample briefly every 2-3 min until the gel slices are completely dissolved!

3. Bind DNA Place a NucleoSpin® Extract II Column into a 2 ml NucleoSpin Collecting Tube and load the sample. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin® Extract II Column back into the collecting tube.

4. Wash silica membrane Add 600 µl buffer NT3. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin® Extract II Column back into the collecting tube.

5. Dry silica membrane Centrifuge for 2 min at 11,000 x g to remove buffer NT3 quantitatively. Make sure the spin column doesn’t come in contact with the flow- through while removing it from the centrifuge and the collecting tube. Residual ethanol from buffer NT3 might inhibit subsequent reactions and has to be removed in this step. In addition to centrifugation, total removal can be achieved by incubation of NucleoSpin® Extract II Columns for 2-5 min at 70°C prior to elution.

6. Elute DNA Place the NucleoSpin® Extract II Column into a clean 1.5 ml microcentrifuge tube. Add 15-50 µl elution buffer NE and incubate at room temperature for 1 min to increase the yield of eluted DNA. Centrifuge for 1 min at 11,000 x g. Yield of larger fragments (>5-10 kb) can be increased by using prewarmed elution buffer (70°C): For elution, add prewarmed elution buffer and incubate at room temperature for 1 min before collecting eluate by centrifugation.

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Concatenation of the Cys-loop receptors ______

C3. Promega Wizard® Plus Minipreps DNA Purification System Vacuum Protocol

Production of Cleared Lysate

1. Pellet 1-10ml overnight culture for 5 minutes. 2. Thoroughly resuspend pellet with 250µl Cell Resuspension Solution. 3. Add 250µl Cell Lysis Solution to each sample; invert 4 times to mix. 4. Add 10µl Alkaline Protease Solution; invert 4 times to mix. Incubate 5 minutes at room temperature. 5. Add 350µl Neutralization Solution; invert 4 times to mix. 6. Centrifuge at top speed for 10 minutes at room temperature.

Binding of Plasmid DNA

7. Attach Vacuum Adapter to manifold port and insert Spin Column into Adapter. 8. Decant cleared lysate into column. 9. Apply vacuum to pull liquid through column. Release vacuum when all liquid has passed through column.

Washing

10. Add 750 µl Wash Solution (ethanol added). Apply vacuum to pull solution through column. 11. Turn off vacuum and repeat Step 10 with 250µl Wash Solution. 12. Dry by applying a vacuum for 10 minutes. 13. Transfer the column to a 2ml Collection Tube and centrifuge at top speed for 2 minutes

Elution

14. Transfer column to a sterile 1.5ml microcentrifuge tube. 15. Add 100µl of Nuclease-Free Water to the column. Centrifuge at top speed for 1 minute at room temperature. 16. 16. Discard column. Store DNA at -20°C or below.

C4. Qiagen HiSpeed® Plasmid Purification Midi Kit Protocol

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2-5 ml LB medium containing the appropriate selective antibiotic. Incubate for approx. 8 hours at 37°C with vigorous shaking (approx. 300 rpm). Use a tube or flask with a volume of at least 4 times the volume of the culture.

2. Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy plasmids inoculate 50 ml medium. For low-copy plasmids, inoculate 150 ml medium. Grow at 37°C for 12-16 h with vigorous shaking (approx. 300 rpm). Use a flask or a vessel with a volume of at least 4 times the volume of the culture. The culture should reach a cell density of approximately 3-4 x 109 cells per millilitre, which typically corresponds to a pellet wet weight of approx. 3 g/liter.

3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C. Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.

4. Resuspend the bacterial pellet in 6 ml Buffer P1. For efficient lysis it is important to use a vessel that is large enough to allow complete mixing of the lysis buffers. Ensure that RNase A has been added to Buffer P1. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle before use to ensure LysBlue particles are completely resuspended. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

5. Add 6 ml Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4-6 times, and incubate at room temperature (15-25°C) for 5 min. Do not vortex, as this will result in shearing of genomic DNA. The lysate should appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still available, continue mixing the solution until a homogeneously colored suspension is achieved. During the incubation prepare the QIAfilter Cartridge: Screw the cap onto the outlet nozzle of the QIAfilter Midi Cartridge. Place the QIAfilter Cartridge into a convenient tube or QIArack.

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6. Add 6ml chilled Buffer P3 to the lysate, and mix immediately and thoroughly by vigorously inverting 4-6 times. Proceed directly to step 7. Do not incubate the lysate on ice. Precipitation is enhanced by using chilled Buffer P3. After addition of Buffer P3, a fluffy white precipitate containing genomic DNA, proteins, cell debris, and KDS becomes visible. The buffers must be mixed completely. If the mixture appears still viscous and brownish, more mixing is required to completely neutralize the solution. It is important to transfer the lysate into the QIAfilter Cartridge immediately in order to prevent later disruption of the precipitate layer. If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.

7. Pour the lysate into the barrel of the QIAfilter Cartridge. Incubate at room temperature for 10 min. Do not insert the plunger! Important: This 10 min incubation at room temperature is essential for optimal performance of the QIAfilter Cartridge. Do not agitate the QIAfilter Cartridge during this time. A precipitate containing proteins, genomic DNA, and detergent will float and form a layer on top of the solution. This ensures convenient filtration without clogging. If, after the 10 min incubation, the precipitate has not floated to the top of the solution, carefully run a sterile pipet tip around the walls of the cartridge to dislodge it. 8. Equilibrate a HiSpeed Midi Tip by applying 4 ml Buffer QBT and allow the column to empty by gravity flow. Flow of buffer will begin automatically by reduction in surface tension due to the presence of detergent in the equilibration buffer. Allow the HiSpeed Tip to drain completely.

9. Remove the cap from the QIAfilter outlet nozzle. Gently insert the plunger into the QIAfilter Midi Cartridge and filter the cell lysate into the previously equilibrated HiSpeed Tip. Filter until all of the lysate has passed through the QIAfilter Cartridge, but do not apply extreme force. Approximately 15 ml of the lysate is generally recovered after filtration.

10. Allow the cleared lysate to enter the resin by gravity flow.

11. Wash the HiSpeed Midi Tip with 20 ml Buffer QC. Allow Buffer QC to move through the HiSpeed Tip by gravity flow.

12. Elute DNA with 5 ml Buffer QF. Collect the eluate in a tube with a minimum capacity of 10 ml.

13. Precipitate DNA by adding 3,5 ml room-temperature isopropanol to the eluted DNA. Mix and incubate at room temperature for 5 min. All solutions should be at room temperature in order to minimize salt precipitation.

14. During the incubation remove the plunger from a 20 ml syringe and attach the QIAprecipitator Midi Module onto the outlet nozzle. Do not use excessive force, bending, or twisting to attach the QIA precipitator! Important: Always remove the QIAprecipitator from the syringe before pulling up the plunger!

15. Place the QIAprecipitator over a waste bottle, transfer the eluate/isopropanol mixture into the 20 ml syringe, and insert the plunger. Filter the eluate/isopropanol mixture through the QIAprecipitator using constant pressure.

16. Remove the QIAprecipitator from the 20 ml syringe and pull out the plunger. Re-attach the QIAprecipitator and add 2 ml 70% ethanol to the syringe. Wash the DNA by inserting the plunger and pressing the ethanol through the QIAprecipitator using constant pressure.

17. Remove the QIAprecipitator from the 20 ml syringe and pull out the plunger. Attach the QIAprecipitator to the 20 ml syringe again, insert the plunger, and dry the membrane by pressing air through the QIAprecipitator quickly and forcefully. Repeat this step.

18. Dry the outlet nozzle of the QIAprecipitator with absorbent paper to prevent ethanol carryover.

19. Remove the plunger from a new 5 ml syringe and attach the QIAprecipitator onto the outlet nozzle. Hold the outlet of the QIAprecipitator over a 1,5 ml collection tube. Add 1 ml of Buffer TE to the 5 ml syringe. Insert the plunger and elute the DNA into the collection tube using constant pressure. Ensure that the outlet of the QIA precipitator is held over the collection tube when Buffer TE ispoured into the syringe, as eluate can drip through the QIAprecipitator before the syringe barrel is inserted. Be careful, as residual elution buffer in the QIAprecipitator tends to foam when expelled. Note: TE Buffer contains EDTA which may inhibit downstream enzymatic or sequencing reactions. Note: Store DNA at -20°C when eluted with water as DNA may degrade in the absence of buffering or chelating agents. 20. Remove the QIAprecipitator from the 5 ml syringe, pull out the plunger and reattach the QIAprecipitator to the 5 ml syringe.

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Concatenation of the Cys-loop receptors ______

21. Transfer the eluate from step 19 to the 5 ml syringe and elute for a second time into the same 1,5 ml tube. This re-elution step ensures that the maximum amount of DNA in the QIAprecipitator is solubilised and recovered.

C5. Invitrogen™ Zero Blunt® TOPO® PCR Cloning Reaction and chemical transformation of One Shot® Competent Cells

Performing the TOPO®Cloning Reaction

Reagent Chemically Competent E. coli Fresh PCR product 0.5 to 4 µl Salt Solution 1 µl Sterile Water Add to a final volume of 5 µl pCR®II-Blunt-TOPO® 1 µl Final Volume 6 µl

1. Mix reaction gently and incubate for 5 minutes at room temperature (22-23°C). Note: For most applications, 5 minutes will yield plenty of colonies for analysis. Depending on your needs, the length of the TOPO® Cloning reaction can be varied from 30 seconds to 30 mintes. For routine subcloning of PCR products, 30 seconds may be sufficient. For large PCR products (> 1 kb) or if you are TOPO® Cloning a pool of PCR products, increasing the reaction time will yield more colonies.

2. Place the reaction on ice and proceed to “Transforming One Shot® Competent Cells”. Note: You may store the TOPO® Cloning reaction at -20°C overnight.

One Shot® Chemical Transformation

1. Add 2 µl of the TOPO® Cloning reaction from “Performing the TOPO® Cloning Reaction” into a vial of One Shot® Chemically Competent E. Coli and mix gently. Do not mix by pipetting up and down.

2. Incubate on ice for 5 to 30 minutes. Note: Longer incubations in ice do not seem to have any effect on transformation efficiency. The length of the incubation is at the user’s discretion.

3. Heat-shock the cells for 30 seconds at 42°C without shaking.

4. Immediately transfer the tubes to ice.

5. Add 250 µl of room temperature S.O.C. medium.

6. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for 1 hour.

7. Spread 10-50 µl from each transformation on a prewarmed selective plate and incubate overnight at 37°C. To ensure even spreading of small volumes, add 20 µl of S.O.C. medium. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies. Incubate overnight at 37°C.

8. An efficient TOPO® Cloning reaction will produce several hundred colonies. Pick 10 colonies for analysis

C6. USB Affymetrix Shrimp Alkaline Phosphatase Tested User Friendly™ Protocol for Dephosphorylation of 5’-ends of DNA in Restriction Enzyme Reaction

1. Digest 1-5 μg of plasmid DNA in a 20 μl volume according to the following table:

DNA > 1 μl 10X Restriction Enzyme Buffer 2 μl Water, Nuclease-Free up to 19 μl Restriction Endonuclease 1 μl

Note: Scale larger reaction volumes proportionally.

2. Incubate at 37°C for 60 min.

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3. Add 1 unit of SAP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and incubate at 37°C for 30-60 min.

4. Stop reaction by heating at 65°C for 15 min. This completely inactivates SAP. Note: Some restriction enzymes require 80°C for complete heat- inactivation. Follow manufacturers’ recommendations.

C7. Ambion mMessage mMachine T7 Transcription Kit Protocol C: Capped Transcription Reaction Assembly

1. Thaw the frozen reagents Place the RNA Polymerase Enzyme Mix on ice, it is stored in glycerol and will not be frozen at –20°C.Vortex the 10X Reaction Buffer and the 2X NTP/CAP until they are completely in solution. Once thawed, store the ribonucleotides (2X NTP/CAP) on ice, but keep the 10X Reaction Buffer at room temperature while assembling the reaction. All reagents should be microfuged briefly before opening to prevent lossand/or contamination of material that may be present around the rim of the tube.

2. Assemble transcription reaction at room temp. The spermidine in the 10X Reaction Buffer can coprecipitate the template DNA if the reaction is assembled on ice. Add the 10X Reaction Buffer after the water and the ribonucleotides are already in the tube. The following amounts are for a single 20 μL reaction. (Reactions was scaled up to 10 μg template DNA) .

Amount Component To 20 μL Nuclease-free water 10 μL 2X NTP/CAP 2 μL 10X Reaction Buffer 0.1-1 μg Linear template DNA 2 μL Enzyme mix

3. Mix thoroughly Gently flick the tube or pipette the mixture up and down gently, and then microfuge tube briefly to collect the reaction mixture at the bottom of the tube.

4. Incubate at 37°C, 1 hr. Typically, 80% yield is achieved after a 1 hr incubation. For maximum yield, we recommend a 2 hr incubation. Since SP6 reactions are somewhat slower than T3 and T7 reactions, they especially may benefit from the second hour of incubation.

5. (optional) Add 1 μL TURBO DNase, mix well and incubate 15 min at 37°C. This DNase treatment removes the template DNA. For many applications it may not be necessary because the template DNA will be present at a very low concentration relative to the RNA. a. Add 1 μL TURBO DNase, and mix well. b. Incubate at 37°C for 15 min.

C8. Qiagen RNeasy Mini Kit RNA Cleanup Protocol

1. Adjust the sample to a volume of 100 μl with RNase-free water. Add 350 μl Buffer RLT, and mix well.

2. Add 250 μl ethanol (96–100%) to the diluted RNA, and mix well by pipetting. Do not centrifuge. Proceed immediately to step 3.

3. Transfer the sample (700 μl) to an RNeasy Mini spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at 8000 x g (10,000 rpm). Discard the flow-through. Reuse the collection tube in step 4. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely..

4. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at _8000 x g (_10,000 rpm) to wash the spin column membrane. Discard the flow-through. Reuse the collection tube in step 5. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added toBuffer RPE before use .

5. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at _8000 x g (_10,000 rpm) to wash the spin column membrane. The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.

6. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min. Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow- through remains on the outside of the RNeasy spin column after step 5.

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7. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30–50 μl RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at _8000 x g (_10,000 rpm) to elute the RNA.

8. If the expected RNA yield is >30 μg, repeat step 7 using another 30–50 μl RNasefree water, or using the eluate from step 7 (if high RNA concentration is required). Reuse the collection tube from step 7. If using the eluate from step 7, the RNA yield will be 15–30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.

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Concatenation of the Cys-loop receptors ______APPENDIX D: DRUGS & SOLUTIONS

D1. MOLECULAR BIOLOGY

DNA loading buffer Compound Amount/volume 0.1% Bromophenol blue 0.1 g 0.1% Xylene cyanol 0.1 g 30% Glycerol 30 ml

ddH2O ad 100 ml Store at 4°C

TE buffer pH 8.0 Compound Amount/volume Tris/HCl (1 M) 100 ml EDTA (1 M) 10 ml

ddH2O ad 1 l Autoclaved

DEPC Compound Amount/volume Diethyl pyrocarbonate 0.1%

ddH2O ad 1 l Autoclaved

TBE buffer Compound Amount/volume TRIS base 54g Boric acid 27.5 g EDTA (0.5 M) 10 ml

ddH2O ad 1 l

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Concatenation of the Cys-loop receptors ______

D2. ELECTROPHYSIOLOGY – CHO CELL MEASUREMENTS

Extracellular solution (bath): Na-Ringer Compound Concentration (mM) [NaCl] 140 [Na-HEPES] 10 [KCl] 4

[CaCl2] 2

[MgCl2] 1 pH adjust. 7.4 adjusted with NaOH/HCl

Intracellular solution (pipette): K-Ringer Compound Concentration (mM) [Na-HEPES] 10 [EGTA] 10 [KCl] 120 [KOH] 31

[MgCl2] 1,785 pH adjust. 7.4 adjusted with KOH/HCl

Na-Ringer pH 6.0 (activation of ASIC1A+BR) Compound Concentration (mM) [NaCl] 140 [Na-HEPES] 5 [MES] 5 [KCl] 4

[CaCl2] 2

[MgCl2] 1 pH adjust. 6.0 adjusted with HCl

GABA was diluted from stock solution in the extracellular Na-Ringer on the day it was used.

All chemicals were purchased from Sigma-Aldrich.

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Concatenation of the Cys-loop receptors ______

D3. ELECTROPHYSIOLOGY – XO MEASUREMENTS

Extracellular solution: Oocyte Ringer (OR-2) Compound Concentration (mM) [NaCl] 90 [Na-HEPES] 5 [KCl] 2.5

[CaCl2] 2.5

[MgCl2] 1 pH adjust. 7.4 adjusted with NaOH/HCl

Pipette solution: 1 M KCl

Low Calcium Barth’s Solution (LBA) Compound Concentration (mM) [NaCl] 90

[NaNO3] 0.66 [KCl] 1

[NaHCO3] 2.4

[MgCl2] 0.82 [Na-HEPES] 10 Gentamicin 0.1 g/L pH adjust. 7.55 adjusted with NaOH/HCl The solution was filtered through a 0.22 m filter.

Modified Calcium Barth’s Solution (MBA) Compound Concentration (mM) [NaCl] 90

[NaNO3] 0.66 [KCl] 1

[NaHCO3] 2.4

[MgCl2] 0.82

[CaCl2] 0.74 [Na-HEPES] 10 Gentamicin 0.1 g/L pH adjust. 7.55 adjusted with NaOH/HCl The solution was filtered through a 0.22 m filter.

GABA and diazepam were diluted from stock solution in OR-2 on the day they were used.

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