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Mechanisms underlying the epileptic phenotype associated with the alpha-1 A322D mutation in the GABA-A… Bradley, Clarrisa Ann 2010

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MECHANISMS UNDERLYING THE EPILEPTIC PHENOTYPE ASSOCIATED WITH THE ALPHA-1 A322D MUTATION IN THE GABA-A RECEPTOR  by  CLARRISA ANN BRADLEY B.Sc., University of Victoria, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2010 © Clarrisa Ann Bradley, 2010  ABSTRACT Epilepsy is a common neurological disorder with a strong hereditary component. A mutation in the α1 subunit (A322D) of the GABA-A receptor is responsible for juvenile myoclonic epilepsy in a large Canadian family. Previous work has identified that this mutation affects the function of GABA-A receptors, expressed in HEK293 cells. Here I have examined the underlying mechanisms of this dysfunction and shown that the mutation reduces the cell surface expression of the GABA-A receptor, promotes association with the endoplasmic reticulum chaperone calnexin, enhances degradation and accelerates the degradation rate of the subunits approximately 2.5 fold. I have also found that the mutation causes the receptor to be degraded by a lysosomal-dependent process. Furthermore, I find that the mutation results in receptors that are inserted into the plasma membrane but are more rapidly endocytosed by a dynamin and caveolin-dependent mechanism. These results suggest that the mutant subunit can form traffickingcompetent receptors that have a shorter lifetime on the plasma membrane. Collectively, my results strongly implicate defects in both the biogenesis and trafficking of the GABAA receptors, as part of the mechanistic basis for the epileptic phenotype observed.  ii  TABLE OF CONTENTS ABSTRACT....................................................................................................................... ii TABLE OF CONTENTS ................................................................................................ iii LIST OF FIGURES ....................................................................................................... viii LIST OF ABBREVIATIONS ......................................................................................... xi ACKNOWLEDGEMENTS .......................................................................................... xiv DEDICATION................................................................................................................. xv  1 INTRODUCTION......................................................................................................... 1 1.1 Epilepsy.................................................................................................................... 1 1.2 The genetics of idiopathic epilepsies ....................................................................... 2 1.3 Mutations associated with epilepsies in the inhibitory system ................................. 4 1.4 GABA receptors....................................................................................................... 5 1.5 GABA-A receptors .................................................................................................. 6 1.6 GABA-A receptor structure and biosynthesis ......................................................... 9 1.7 GABA-A receptor oligomerization and assembly................................................. 11 1.8 Endoplasmic reticulum chaperones and GABA-A receptor.................................. 12 1.9 ER-associated degradation..................................................................................... 14 1.9.1 Recognition and export of misfolded glycoproteins....................................... 14 1.9.2 Ubiquitination of misfolded glycoproteins for ERAD.................................... 19 1.9.3 Retrotranslocation and proteasomal degradation of misfolded glycoproteins................................................................................................. 21 1.10 Degradation mechanisms of GABA receptors..................................................... 22 iii  1.11 GABA-A receptor interactors and cell surface trafficking .................................. 25 1.12 Rationale, hypothesis and objectives ................................................................... 28  2 EXPERIMENTAL METHODS ................................................................................ 30 2.1 Cell culture and plasmids....................................................................................... 30 2.2 Transient transfections of HEK293 cells ............................................................... 31 2.3 Cell ELISA assay for surface receptor expression levels ...................................... 32 2.4 Immunocytochemistry ........................................................................................... 34 2.4.1 Cell surface and intracellular expression immunocytochemistry ................... 35 2.4.2 Colocalization of GABA-A receptors and early endosomes .......................... 35 2.5 Western blotting..................................................................................................... 37 2.5.1 HEK293 sample preparation for western blotting .......................................... 37 2.5.2 Denaturing polyacrylamide gel electrophoresis.............................................. 38 2.5.3 Western blotting method................................................................................. 38 2.5.4 Western blotting antibodies and primary antibody conditions. ...................... 39 2.6 Coimmunoprecipitation assays .............................................................................. 40 2.6.1 Examination of transfected α1 binding with endogenous calnexin ................ 40 2.6.2 Coimmunoprecipitation of the GABA-A receptor α1 subunit:caveolin 1 protein complex in transfected HEK293 cells ................................................ 41 2.7 Metabolic pulse-chase assays ................................................................................ 42 2.8 Treatment conditions ............................................................................................. 43 2.8.1 Degradation blockade assay conditions .......................................................... 43 2.8.2 Protein folding assay conditions ..................................................................... 44  iv  2.8.3 Dynamin and caveolin blockade conditions ................................................... 44 2.8.4 N-glycosylation blockade with 35S-methionine labeling ................................ 45 2.9 Surface biotinylation assays................................................................................... 45 2.10 Amino acid sequence alignment .......................................................................... 46 2.11 Statistical analysis................................................................................................ 47  3 RESULTS .................................................................................................................... 48 3.1 Introduction............................................................................................................. 48 3.2 Results.................................................................................................................... 49 3.2.1 The effects of the 1(A322D) mutation on the cell surface expression of GABA-A receptors expressed in HEK293 cells......................................... 49 3.2.2 The effects of the 1(A322D) mutation on total expression of GABA-A receptors expressed in HEK293 cells. ........................................................... 52 3.2.2 The A322D mutation causes greater cell stress upon expression in HEK293 cells and neurons.............................................................................. 57 3.2.4 The A322D mutation causes enhanced association with the ER chaperone calnexin.......................................................................................... 60 3.2.5 Lowering the incubation temperature does not affect the expression of mutant α1 containing GABA-A receptors .................................................................. 72 3.2.6 A chemical chaperone does not significantly rescue the expression level of mutant containing GABA-A receptors. ............................................. 75 3.2.7 The A322D mutation enhances the degradation rate of GABA-A receptors......................................................................................................... 78  v  3.2.8 Pathways involved in the degradation of wild type and mutant α1 subunits ........................................................................................................... 87 3.2.9 The early endosome marker Rab4 colocalizes with A322D mutant containing GABA-A receptors ....................................................................... 95 3.2.10 Blocking dynamin-dependent endocytosis enhances the total expression of mutant GABA-A receptors..................................................... 98 3.2.11 The cell surface expression of mutant GABA-A receptors is also increased after blockade of dynamin-dependent endocytosis....................... 99 3.2.12 The total expression of mutant GABA-A receptors is increased after blockade of caveolin 1 ............................................................................... 102 3.2.13 The cell surface expression of mutant GABA-A receptors is also increased after blockade of caveolin-dependent endocytosis ..................... 108 3.2.14 A novel endocytosis pathway for the internalization of GABA-A receptors..................................................................................................... 111  4 DISCUSSION ............................................................................................................ 116 4.1 GABA-A receptor mutations that are associated with an epileptic phenotype ... 116 4.2 Summary of the results ........................................................................................ 119 4.3 Changes in the surface expression of the α1(A322D) mutant ............................. 120 4.4 Changes in total expression of the α1(A322D) mutant........................................ 121 4.5 The mutant receptor shows enhanced misfolding................................................ 121 4.6 Effects of temperature and DMSO on the expression of mutant GABA-A receptors .............................................................................................................. 123  vi  4.7 Protein degradation rates are increased by the 1(A322D) mutation. ................ 124 4.8 The mutation is associated with a reduction in glycosylated subunits ................ 126 4.9 The role of ERAD in the degradation of mutant α1(A322D) GABA-A receptors .............................................................................................................. 127 4.10 The role of lysosomal degradation of mutant α1(A322D) GABA-A receptors ............................................................................................................. 129 4.11 The role of dynamin-dependent endocytosis in the trafficking of wild type and mutant GABA-A receptors.......................................................................... 131 4.12 The role of caveolin-dependent endocytosis in the trafficking of wild type and mutant GABA-A receptors.......................................................................... 132 4.13 Evidence for an interaction between GABA-A receptors and caveolin-1......... 136 4.14 Mechanisms contributing to the reduction in GABA-A receptor function in the α1(A322D) mutant. .................................................................................. 138 4.15 Future Directions ............................................................................................... 142 REFERENCES ...........................................................................................................146  vii  LIST OF FIGURES Figure 1.1. Structure of GABA-A-receptors .................................................................... 7 Figure 1.2. Site of action of GABA and some common drugs ........................................ 8 Figure 1.3. Schematic diagram of the ER degradation pathway of misfolded proteins 16 Figure 1.4. Schematic diagram of the GABA-A receptor trafficking and degradation pathways.................................................................................. 23 Figure 1.5. Proteins which interact with various subunits of GABA-A receptors ........ 26 Figure 2.1. Colorimetric cell ELISA assay used to quantify the surface expression of GABA-A receptors .................................................................................. 33 Figure 2.2. Surface and intracellular immunostaining method for GABA-A receptor distribution .................................................................................... 36 Figure 3.1. The α1(A322D) mutation causes a marked reduction in the cell surface expression of GABA-A receptors observed by immunocytochemistry .... 51 Figure 3.2. The A332D mutation results in a reduction in both surface expression and total expression of GABA-A receptors ................................................ 54 Figure 3.3. The A322D mutation leads to reduced total expression of the α1 subunit.. 56 Figure 3.4. The A322D mutation results in a dramatic decrease in the total expression of the GABA-A receptor.......................................................... 59 Figure 3.5. The A322D mutation increases the co-association of GABA-A receptor subunits with the endoplasmic reticulum chaperone calnexin....... 63 Figure 3.6. Expression of the mutant form of the GABA-A receptor does not alter the total amount of calnexin......................................................................... 65  viii  Figure 3.7. The A322D mutation increases the co-association of the α1 subunit with calnexin ................................................................................................ 67 Figure 3.8. The A322D mutation similarly increases the co-association of the α1β2 receptor with calnexin.................................................................................. 69 Figure 3.9. The A322D mutation likewise increases the co-association of the α12 combination with calnexin ........................................................................... 71 Figure 3.10. Low temperature incubation does not alter the ratio of wild type to mutant GABA-A receptor ......................................................................... 74 Figure 3.11. The protein folding chemical chaperone DMSO partially increases the expression levels of mutant containing GABA-A receptors ..................... 77 Figure 3.12. The 1(A322D) mutation reduces the half-life of the α1 subunit............. 81 Figure 3.13. The 1(A322D) mutation reduces the half-life of the full GABA-A receptor complex ....................................................................................... 83 Figure 3.14. Effects of inhibiting N-glycosylation with tunicamycin ........................... 86 Figure 3.15. Total ubiquitination products are increased by lactacystin treatment ....... 89 Figure 3.16. The proteasome inhibitor lactacystin enhances both wild type and mutant containing GABA-A receptor expression ..................................... 91 Figure 3.17. The lysosomal peptidase inhibitor leupeptin preferentially blocks degradation of the α1(A322D) containing GABA-A receptors................. 94 Figure 3.18. Early endosomes appear more abundant, and are colocalized with mutant GABA-A receptors ........................................................................ 97 Figure 3.19. Blocking dynamin-dependent processes enhances the total expression of α1 and α1(A322D) containing receptors ............................................. 101  ix  Figure 3.20. Blocking dynamin-dependent endocytosis preferentially enhances the surface expression of α1(A322D) containing receptors .................... 104 Figure 3.21. Blocking caveolin 1-dependent endocytosis enhances the total expression of α1(A322D) containing receptors ....................................... 107 Figure 3.22. Blocking caveolin 1-dependent endocytosis preferentially enhances the surface expression of α1(A322D) containing receptors .................... 110 Figure 3.23. A putative consensus caveolin binding domain in the rat GABA-A receptor α subunits ................................................................................... 113 Figure 3.24. Co-IP of caveolin 1 and GABA-A receptor α1 subunits expressed in HEK293 cells........................................................................................... 115 Figure 4.1. GABA-A receptor mutations associated with epilepsy. (Modified from Macdonald and Kang, 2009 .............................................................. 117 Figure 4.2. Likely sites of action of the dominant negatives endocytosis constructs used in this study........................................................................................ 135 Figure 4.3. Summary of how the α1(A322D) mutation affects the GABA-A receptor ...................................................................................................... 141  x  LIST OF ABBREVIATIONS G  Gibbs free energy  AChR  acetylcholine receptor  AMPA  α-3-amino -3-hydroxy-5-methylisoxazole-4-propionate  AP2  adaptor protein  ATF6  activating transcription factor 6  BDNF  brain derived neurotrophic factor  BiP  immunoglobulin binding protein  Cav-DN  caveolin 1 dominant negative  Cdc48  cell division cycle 48  CFTR  cystic fibrosis transmembrane conductance regulator  CNS  central nervous system  CNQX  6-cyanonitroquinoxaline-2,3-dione  Co-IP  coimmunoprecipitate  CPM  counts per minute  CSD  caveolin-1 scaffold domain  CUL3  cullin-3  D-AP5  D-2-amino-5-phosphonopentanoate  DMEM  Dulbecco’s modified eagle medium  DMSO  dimethylsulfoxide  D-PBS  Dulbecco’s phosphate buffered saline  ECL  enhanced chemiluminescence  ECS  extracellular solution  EDEMs  ER degradation-enhancing α-mannosidase-like proteins  EDTA  ethylenediaminetetra acetic acid  EEG  electroencephalogram  EGF  epidermal growth factor  EGTA  ethylene glycol bis [b-aminoethylether] N,N,N',N'-tetra acetic acid  ER  endoplasmic reticulum  ERAD  ER-associated degradation  xi  FBS  fetal bovine serum  G  GTP binding  GABA  -aminobutyric acid  GABARAP  GABA-A receptor-associated protein  GEFS+  generalized epilepsy with febrile seizures plus  GRIP  glutamate receptor interacting protein  Grp 94  94-kDa glucose regulated protein  GS  goat serum  HEK293  human embryonic kidney 293  HEPES  4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid  HCMV  Human cytomegalovirus  HECT  Homologous to the E6-AP Carboxyl Terminus  HRP  horseradish peroxidase  Hsp  heat shock protein  IB  immunoblotting  IGE  idiopathic generalized epilepsy  IgG  immunoglobulin G  IP  immunoprecipitation  IRE1  inositol requirement 1  JME  juvenile myoclonic epilepsy  KEL-8  kelch repeat containing protein 8  µCi  microCurie  µg  microgram  µl  microliter  µm  micrometer  MHCI  major histocompatibility complex I  mIPSC  miniature inhibitory postsynaptic current  mM  millimolar  mOsm  milliosmole  ng  nanograms  NSF  N-ethylmaleimide-sensitive factor  xii  OPD  o-phenylenediamine dihydrochloride  PAGE  polyacrylamide gel electrophoresis  PAM  protein associated with myc  PDIs  protein disulfide isomerases  PDL  poly-D lysine  PI3K  phosphoinositide 3-kinase  Plic  proteins that link IAP (integrin associated protein) to the cytoskeleton  PDI  protein disulphide isomerases  PERK  protein kinase-like endoplasmic reticulum kinase  PKB  protein kinase B  Plic-1  protein linking integrin-associated protein to cytoskeleton-1  PM  plasma membrane  PVDF  polyvinylidene difluoride  RIPA  radioimmunoprecipitation assay  RT  room temperature  SDS  sodium dodecyl sulfate  SE  status epilepticus  TBST  Tris-buffered saline + Tween-20  TGF  transforming growth factor beta  TM  transmembrane  TTX  tetrodotoxin  Ub  ubiquitin  UGGT  UDP:glucose:glycoprotein glucosyltransferase  UPR  unfolded protein response  VIMP  Valosin-containing protein-interacting membrane protein  xiii  ACKNOWLEDGEMENTS I wish to thank my family, friends and lab mates who have supported and inspired me throughout this work. In particular, I would like to thank my mother for her unflagging love and support. I am grateful to my partner, Graham, for his constructive criticism of my thesis, always cheerful attitude and bottomless love. Thanks to members past and present for making my time here enjoyable as well as instructive and interesting. It has been a great pleasure to work in this lab! A special thanks to Steve Van Iderstine, Jie Lu, Lidong Lu, Yu Ping Li, Taesup Cho, Yushan Wang and Henry Martin. A very big thank you to Changiz Taghibiglou for sharing his great enthusiasm for science with me. Most of all I’d like to express my gratitude and thanks to my supervisor, Yu Tian Wang, for critical discussion, incisive direction and his consistent, though understated, support along the way. I have learnt a considerable amount about research and life in this lab. I sincerely thank my advisory committee members for their insights and helpful suggestions along the way: Dr Alaa El-Husseini, Dr Lynn Raymond and Dr. Steve Vincent. I especially grieve the loss of Dr El-Husseini who passed away in early 2008. I miss his excellent research advice and supportiveness, and will always remember the kind person that he was.  xiv  DEDICATION I dedicate this work to my son James, whose life began during the course of my write up of this work, and has filled my life with much love and laughter.  xv  CHAPTER 1 INTRODUCTION  1.1 Epilepsy Epilepsy is one of the most common neurological disorders and has a life time incident rate of up to 3% (Hauser et al., 1996; Wisden et al., 1992). It is a chronic neurological disorder that manifests as recurrent and often unprovoked seizures (Robert et al., 2005) and most frequently occurs early on in development. These seizures are a result of aberrant, excessive, and synchronous epileptiform bursts of activity in the brain that is usually detectable by the use of electroencephalogram (EEG) recordings during an epileptic event. Epilepsy is not one but a family of heterogenous disorders. These epilepsy types may be diagnosed dependent on several factors such as patient age, area of the brain affected, mode of propagation and whether the aberrant activity is focal or generalized across brain regions as well as on patient behaviour. Epilepsy may also be “symptomatic” as in the event of a stroke, cortical malformation or lesion. Others may be of unknown cause, or “idiopathic”, because the brain structure shows no particular malformations and no other obvious insults have been determined (Weber and Lerche, 2008). Idiopathic epilepsies represent up to 47% of all epilepsies and are generally believed to have a high genetic component (Freitag et al., 2001).  1  1.2 The genetics of idiopathic epilepsies The most common genetic epilepsies show a complex pattern of inheritance, but the genes involved are largely unknown (Kaneko et al., 2002). Systems that are implicated in the generation of idiopathic epilepsies include defects in neurotransmitter release, vesicle recycling, and membrane excitability as well as altered neurotransmitter receptor function. Interestingly, many of the epilepsy related genes discovered so far are genes that encode ion channels or encode proteins that have been suggested to interact with ion channels and thus, alter the firing balance towards hyperexcitablity (Hirose et al., 2003; Weber and Lerche, 2008). This has led some to believe that most epilepsies are, in fact, channelopathies (Hirose, 2006). Thus far, epilepsies have been linked to mutations in the voltage-gated potassium and sodium channels, the sodium/potassium ATPase pump, GABA-A receptors, chloride channels, acetylcholine receptors, calcium channels, glutamate transporters and glutamate receptors to name a few (Noebels, 2003). However, only about 1-2% of idiopathic epilepsies are monogenic while the remainder are believed to be polygenic (Kaneko et al., 2002; Weber and Lerche, 2008). For example, several naturally occurring mutations in transmembrane domain 2 of the acetylcholine receptor (AChR) α4, α2 and β2 subunits leads to increased acetylcholine sensitivity and has been strongly linked to autosomal dominant nocturnal frontal lobe epilepsy (Aridon et al., 2006; Lerche et al., 2005; Steinlein, 2004; Steinlein et al., 1995). While this epilepsy is quite specific in its symptoms and its related underlying dysfunction, others, such as generalized epilepsy with febrile seizures plus (GEFS+), are more a spectrum of related disorders. Consistent with a broader range of clinical features, GEFS+ also shows more genetic heterogeneity within families and subunits of  2  the voltage gated sodium channel (SCN1A & SCN1B) and GABA-A receptor (GABRG2 and GABRD) have been implicated as causative (Weber and Lerche, 2008). It has been important to look at these unique monogenetic mutations that lead to specific epilepsies as it appears that more complex or more distantly related genetic mutations may be converging to disrupt the same system. For instance, in an epileptic animal model, decreased sodium channel expression of mutant SCN1A mice was observed specifically in GABAergic interneurons (Ogiwara et al., 2007; Yu et al., 2006). These interneurons also showed decreased excitability suggesting that these sodium channel mutations may largely be affecting the inhibitory system (Lerche et al., 2001) and resulting in an epileptic phenotype. Idiopathic generalized epilepsy (IGE) is another complex inheritable form of epilepsy that encompasses a class of disorders rather than one particular type. The most common idiopathic generalized epilepsies (IGEs) include childhood absence epilepsy, juvenile absence epilepsy and juvenile myoclonic epilepsy. As with GEFS+, IGEs have various genes associated with the syndrome (Weber and Lerche, 2008). Mutations in the GABA-A receptor (Cossette et al., 2002; Maljevic et al., 2006) and calcium channel or calcium regulation have been found (Haug et al., 2003; Saint-Martin et al., 2009). Juvenile myoclonic epilepsy (JME) is characterized by awakening myoclonus (muscle twitch or jerking seizures occur within 1 h of awakening), absence seizures associated with fast spike-wave discharges on EEGs, and generalized tonic-clonic seizures (also known as grand mal seizures) (Kaneko et al., 2002). It has been particularly difficult to determine JME’s underlying genetic cause, but it recently has been linked to mutations  3  leading to alterations in calcium turnover, calcium channel mutations and a mutation in the α1 subunit of the GABA-A receptor (Cossette et al., 2002; Weber and Lerche, 2008).  1.3 Mutations associated with epilepsies in the inhibitory system While mutations in several ion channels and receptors of the excitatory system have also been found in association with epilepsy, alteration to GABAergic inhibitory transmission has long been suspected to be involved in epileptogenesis (Noebels, 2003). Indeed, the inhibitory system is the most commonly affected system amongst known genetically based epilepsies (Galanopoulou, 2010; Noebels, 2003). At the molecular level, many mutations have been found to disrupt inhibitory transmission and produce an epileptic phenotype (Noebels, 2003). Examples of some mutations that reduce GABA synthesis have been found in glutamic acid decarboxylase 65 (Kash et al., 1999) one of only two enzymes important in the conversion of glutamate to form GABA, in excitatory amino acid transporter 1 (Sepkuty et al., 2002), a glutamate transporter, and tissue non-specific alkaline phosphatase (Waymire et al., 1995), which reduces pyridoxine-dependent GABA synthesis. Mutations in potassium ion channels such as Kv3.2 (Tansey et al., 2002) and Kv1.1 (van Brederode et al., 2001) may preferentially alter interneuron excitability and thus inhibitory transmission (Noebels, 2003). Mutations in Cav2.1 produce generalized absence seizures and may alter inhibitory presynaptic release more so than at excitatory synapses (Caddick et al., 1999). Mutations in GABA receptors disrupt inhibition in the post-synaptic cell leading to its excessive firing and thus these mutations are strongly associated with a variety of epileptic syndromes (Galanopoulou, 2010; Noebels, 2003)) 4  1.4 GABA receptors The neurotransmitter gamma aminobutyric acid (GABA) is the major inhibitory chemical signal in the vertebrate brain (for review see Cherubini and Conti, 2001; Mehta and Ticku, 1999). Three main types of receptor have been found to bind the GABA ligand, which have been grouped according to structural, pharmacological or physiological properties. GABA-A and GABA-C receptors are postsynaptic ligand gated ion conducting channels, which upon binding GABA allow the influx of chloride ions into the cell resulting in hyperpolorization of adult neurons. GABA-B receptors are heterodimeric metabotropic receptors coupled to heterotrimeric GTP-binding (G) proteins, which belong to the G protein coupled receptor superfamily. GABA-B receptors are expressed both pre- and post-synaptically were they mediate different functions in response to GABA binding. Activation of pre-synaptic GABA-B receptors autoregulates GABA release via depression of Ca2+ influx and thus, inhibits the release of more neurotransmitter at GABAergic synapses (Bowery and Enna, 2000; Davies et al., 1990; Khazipov et al., 1995). Both GABA-A and GABA-B are widely distributed throughout the central nervous system (CNS). However, GABA-C receptors, which are made up of homomeric  subunits (1-3), are largely expressed in the retina. It should be noted, however, that  subunits are expressed in the adult and juvenile rat hippocampus (Liu et al., 2004; Rozzo et al., 2002) where they may mediate paired-pulse depression of inhibitory currents in CA1 pyramidal cells (Xu et al., 2009). However, the distinction between GABA-A and GABA-C receptors may not be quite so clear since  subunits  5  may also combine with GABA-A receptor subunits in the hippocampus (Hartmann et al., 2004) leading some to suggest that  subunits should be classed as a type of GABA-A receptor.  1.5 GABA-A receptors The GABA-A receptor is the predominant mediator of fast synaptic inhibition as well as a low level tonic inhibition in the brain (for reviews see Arancibia-Carcamo and Kittler, 2009; Farrant and Nusser, 2005; Luscher and Keller, 2004; Semyanov, 2003; Tretter and Moss, 2008). The GABA-A receptor is believed to be a heteropentameric protein complex that typically contains two , two  and one  subunit ((Baumann et al., 2002; Chang et al., 1996; Connolly et al., 1999; Gorrie et al., 1997; Tretter et al., 1997) Figure 1.1). In addition, the receptor can be assembled from seven subunit classes with multiple members and splice variants: (1-6), (1-3),  (1-3), , , ,  (Mehta and Ticku, 1999; Wisden et al., 1992). GABA-A receptor function is modulated by a number of various endogenous and pharmacologically relevant drugs (Miller and Smart, 2010) (Figure 1.2). Within the same subunit class amino acid identity is 70%, whereas the identity between classes is 30% (Mehta and Ticku, 1999). The most common receptor found in the brain contains the 1, 2 and 2 subunits (Baumann et al., 2002; Connolly et al., 1999; Davies et al., 1997; Macdonald and Olsen, 1994). This subtype accounts for approximately 60% of the total GABA-A receptor population. In heterologous cells, the minimum subunit  6  Figure 1.1. Structure of GABA-A receptors. A. The GABA-A receptor is comprised of five subunits which all span the plasma membrane. They form an ion channel that is permeable to Cl-. B. Each subunit is a polypeptide of approximately 450 amino acids (blue circles) that span the plasma membrane (grey) four times. The large N-terminal region is glycosylated at various sites and there is a disulphide bond that is characteristic of the cys-cys loop family of ligand-gated ion channels. C. The subunits are arranged such that the second transmembrane regions align to form the pore.  A  B Cl ‐ β  α  γ  glycosylation  N  C C  β α  α  1 2 3  β γ 7  4  Disulphide bond  Figure 1.2. Site of action of GABA and some common drugs.  GABA  Pentobarbital  Diazepam  Allopregnanolone  Ethanol  Isoflurane  Propofol  8  composition required for cell surface expression of the receptor are α and β subunits (Whiting et al., 1999). It is unclear to what extent these receptors exist in neuronal populations, but extrasynaptic αβ receptors have been observed in rat hippocampal pyramidal cells (Mortensen and Smart, 2006). Nonetheless, based on a comparison of recombinant receptors with native receptor pharmacology and channel properties, the majority of native GABA-A receptors likely contains the 2 subunit. Replacement of the 2 subunit with any of the , , ,  subunits confers considerably more heterogeneity to the GABA-A receptor population resulting in different pharmacological properties, channel gating and agonist affinity characteristics (Sieghart and Sperk, 2002; Whiting et al., 1999). Indeed, possible subunit combinations have been estimated to be upwards of 500 different types of receptors where α and β subunits may be of different class members (Sieghart and Sperk, 2002). This estimate may be quite conservative, even when observing the spatio-temporal expression of the subunits as well as assembly rules, because small subpopulations of GABA-A receptors are difficult to isolate using current methods (Caspary et al., 1999; Chang et al., 1995; De Blas, 1996; Sieghart and Sperk, 2002).  1.6 GABA-A receptor structure and biosynthesis GABA-A receptors belong to the evolutionarily conserved heteropentameric nicotinic superfamily of ligand gated ion channels, which include the nicotinic acetylcholine, glycine and 5-hydroxytryptamine 3 receptors (Barnard et al., 1998; Ortells and Lunt, 1995). GABA-A receptor subunits have a long extracellular N terminal  9  domain, four transmembrane (TM) regions, an intracellular loop between TM 3 and TM 4, and a short extracellular C terminus (Tretter and Moss, 2008; Figure 1.1B). Transmembrane 2 is believed to form the pore lining side of the receptor (Figure 1.1C). The vast majority of what is known about the biosynthesis of these receptors has been derived from experiments performed in heterologous cell lines. Based on these studies, the general principles of GABA-A receptor subunit motif interactions, receptor stochiometery and subunit oligomerization have been determined. These rules have been shown to likewise govern the formation of these receptors in their native neuronal environment. Proteins bound for secretion or membrane insertion, such as GABA-A receptors, are synthesized in the endoplasmic reticulum (ER). The mRNA bound ribosome attaches via the signal recognition particle receptor and the ribosome receptor to the ER after initial synthesis of the leader sequence containing an ER localizing signal. The nascent polypeptide chain is elongated through the sec61 translocon pore which spans the ER membrane. In the case of multi-membrane spanning proteins, the newly synthesizing protein begins to traverses the ER membrane several times through a series of stop and start sequences at either end of the transmembrane domains. N-linked glycosylation of the nascent polypeptide occurs as a large branched chain of three glucose, nine mannose and two N-acetylglucosamines are transferred to the asparagine residue upon recognition of the N-X-S/T motif (where X is any amino acid except proline) by a membrane bound oligosaccharyl transferase enzyme (Helenius and Aebi, 2004). This occurs co-translationally as the N-terminus of the GABA-A receptor subunits are synthesized. N-linked glycosylation of proteins in the ER plays an important  10  role in protein folding, oligomerization, quality control, sorting and transport (Helenius and Aebi, 2001). There are two known N-linked glycosylation sites at positions 10 & 110 in the α1 subunit (Buller et al., 1994); Figure 1.1.B). Analysis of the β2 and 2 long amino acid sequences (Connolly et al., 1996) also showed two consensus sites for the β2 subunit (at positions 8 and 80) and three sites for the 2 long subunit (at positions 13, 90 and 208). Thus, treatment of HEK293 cells expressing each of these subunits with tunicamycin and pulse labeled with 35S-methionine showed an appropriate reduction of the large molecular weight species for all three subunits to single bands. Indeed, N-glycosylation for GABA-A receptors appears to be a critical factor in the functional expression of these receptors. Point mutations of GABA-A receptor α1 subunit in the Asn-X-Ser/Thr sites have shown reduced whole cell currents of recombinant receptors expressed in Xenopus laevis (Buller et al., 1994).  1.7 GABA-A receptor oligomerization and assembly Assembly of GABA-A receptors occurs independently of N-linked glycosylation of the subunits and may occur as early as 5 min after translation in the ER (Connolly et al., 1996; Helenius and Aebi, 2001). Both trafficking (α1β2) and non-trafficking (α12 and β22) subunit combinations are capable of oligomerizing and can coimmunoprecipitate (co-IP) together upon expression in HEK293 cells. In general, homomeric subunits can oligomerize, but do not reach the cell surface. Assembly sequences have been found in the N-terminus of α1, β2/3, 2/3 subunits (see review Sarto-Jackson and Sieghart, 2008) clustering within amino acids 40 to 180, which govern the assembly of receptors and  11  restrict the number of interactions and types of receptors formed (Bollan et al., 2003; Klausberger et al., 2001; Klausberger et al., 2000; Taylor et al., 2000; Taylor et al., 1999). Indeed, certain regions and amino acids have been shown to give specificity to the type of subunit the other can bind. For example, the assembly cluster RQS (66-68 amino acids) in the α1 subunit can bind both β1 and β3 subunits, but excludes β2 (Bollan et al., 2003). However, mutation of R66 allows β2 to also bind. In addition, there are also amino acids or sequences in these assembly boxes which can, when mutated, allow homomeric receptors to form. Two such sequences have been found and both allow homomeric β3 (GKER sequence) or, upon point mutation (H109C) allows homomeric α1 to form. Interestingly, both are also expressed on the cell surface in heterologous cell lines (Sarto-Jackson et al., 2007; Taylor et al., 1999). From the use of heterologous cell lines and expression of single subunits as well as concatamers to reconstruct the electrophysiological properties of the native GABA-A receptor, the stoichometry and the arrangement of the receptor has been determined to be -β-α-β-α (Baumann et al., 2002; Tretter et al., 1997). Under normal conditions the αβ and αβ represent surface competent receptor types which exist while α combinations are retained in the ER and do not reach the cell surface (Connolly et al., 1996).  1.8 Endoplasmic reticulum chaperones and GABA-A receptor A large number of molecular chaperones exist in the cytosol and organelles of the cell. These include the heat shock family of proteins, for example heat shock protein (Hsp) 60 and 70, and others such as protein disulphide isomerases, calnexin, and  12  calreticulin (Helenius and Aebi, 2004; Kleizen and Braakman, 2004; Vembar and Brodsky, 2008). It has been demonstrated by cross-linking and co-IP experiments that chaperones work in protein complexes and often have co-chaperones such as Hsp 40. In the ER, a variety of proteins act upon the nascent protein to remove and attach complex carbohydrates and assist in proper folding. As with most N-linked glycoproteins, GABA-A receptors have been found to co-IP with both calnexin and with immunoglobulin binding protein (BiP) chaperones (Connolly et al., 1996). Although other chaperone type interactions may also exist, these are the two major molecular chaperones for transmembrane glycoproteins bound for the cell surface. When both are needed for folding of a particular protein, calnexin and BiP act sequentially. Indeed, these chaperones have not been found in complexes together such that if calnexin is bound then BiP is not (Kleizen and Braakman, 2004; Molinari and Helenius, 2000). BiP (Hsp70 family), which binds to stretches of exposed hydrophobic domains in unfolded proteins are luminal chaperones (Johnston and Madura, 2004). In the native folded state hydrophobic domains are buried in the interior of soluble proteins or associated with membrane lipids in membrane bound proteins to maintain the lowest energy state. Exposure of these domains can lead to inappropriate interactions and aggregation. BiP, along with its co-chaperones J domain containing Hsp40 family of proteins and dedicated nucleotide-exchange factors regulate the solubility of folding proteins (Knittler et al., 1995; Schmitz et al., 1995). ATP bound BiP binds Hsp40s with a polypeptide substrate. The folding peptide is transferred to BiP and the Hsp40 is released. Hydrolysis of ATP to ADP bound to BiP recruits nucleotide exchange factors, which in turn catalyzes the release of the correctly folded peptide and BiP. If the protein  13  has not reached its proper conformation, BiP also has a role in activating the mechanism that helps misfolded proteins become ERAD substrates. In addition, the ER contains the lectin chaperones, calnexin and calreticulin, which bind to the N-glycosylated sugar moieties to aid in proper protein folding (Helenius and Aebi, 2004). This is done through an indirect tagging system in which glucosidases I and II trim the sugar moieties attached to the nascent polypeptide. Glucose trimming by glucosidase II creates binding sites for the membrane bound calnexin or the soluble ER luminal calreticulin. Proteins which are undergoing folding are cycled through deglycosylation, reglycosylation and calnexin/calreticulin binding. This cyclical process prevents aggregation and export of incompletely folded chains. Upon completion of the nascent chains, however, the protein will undergo one of three fates. If appropriately folded, it leaves the ER. If it is incompletely folded (showing hydrophobic patches or is in a molten globule-like state) the protein is monoglycosylated by UDP:glucose:glycoprotein glucosyltransferase (UGGT) (Caramelo et al., 2003; Taylor et al., 2003). They are then rebound by calnexin/calreticulin in further attempts to achieve its appropriate folding state. Lastly, it may be degraded by ER-associated degradation (ERAD) via retrotranslocation likely through the translocon complex.  1.9 ER-associated degradation  1.9.1 Recognition and export of misfolded glycoproteins  ERAD is a critical mechanism by which the cell prevents the accumulation of unsalvageable misfolded or misassembled glycoproteins (Helenius and Aebi, 2004;  14  Vembar and Brodsky, 2008). ERAD is the translocation of newly synthesized proteins for degradation by cytoplasmic proteasomes (Johnston and Madura, 2004; Kostova and Wolf, 2003). The unfolded protein response (UPR) is a mechanism that detects damaged proteins and assists in blocking the accumulation of aggresomes; a cell lethal event. This is largely dependent on the chaperone BiP for activation and has been reviewed extensively (Rutkowski and Kaufman, 2004). In brief, the UPR is initiated by the titration away of BiP from the UPR sensor molecules resident in the ER membrane PERK, IRE1 and ATF6 to bind misfolding proteins. Together, these sensor molecules initiate a time dependent cascade of events that acts to inhibit general protein synthesis, increase transcription of ER chaperones such as BiP, PDI and Grp 94 and ERAD degradation genes such as EDEM (Yoshida et al., 2003). These two mechanisms may be activated together under ER stress so that the protein burden in the ER is considerably reduced. Broadly speaking, there are three stages by which ERAD removes unwanted glycoproteins: substrate recognition of a misfolded / misassembled / unassembled glycoprotein, cytoplasmic retrotranslocation, and finally proteasomal degradation (Vembar and Brodsky, 2008; Figure 1.3). It is not entirely clear how the ERAD machinery recognizes proteins which have misfolded versus those that are incompletely folded. It seems that a large percentage of “wild type” proteins, such as CFTR, are destroyed by ERAD and it has also been suggested to be a means of regulating the numbers of available proteins for membrane insertion (Turnbull et al., 2007). Thus, many proteins that may appear questionable to the quality control machinery become  15  Figure 1.3. Schematic diagram of the ER degradation pathway of misfolded proteins.  cytosolic proteins  misfolded proteins  ER lumen  cytosol membrane proteins  ubiquitin ligase  Recognition 26S proteasome  Ub chain Cdc48p/p97 complex Rad23p/DSK2p  Retrotranslocation and ubiquitination  Degradation  16  targets of ERAD. The distinction between misfolded proteins and true folding intermediates appears to be largely dependent on time. Misfolded glycoproteins start to degrade between 30-90 min after synthesis by exponential decay (Helenius and Aebi, 2004) in mammalian cells. The lag in degradation of misfolded proteins appears to be dependent on modification of the glycoprotein by mannosidases such as α-1,2 exomannosidase in yeast and ER mannosidase I in mammals, which specifically trims the terminal mannose to yield the Man8GlcNAc2 B-isomer (Herscovics, 2001). If these mannosidases are pharmacologically inhibited or mutated, glycoprotein degradation is dramatically slowed (Jakob et al., 1998; Liu et al., 1999). Likewise with overexpression of a mannosidase, ERAD is dramatically upregulated (Wu et al., 2003). These relatively slow acting enzymes allow sufficient time for nascent peptides to reach their appropriate conformational state (Helenius and Aebi, 2004). Lag times for mannose removal in individual proteins can vary from 10 min in yeast to 1 h in mammalian cells and time until initiation may also be dependent on the number of glycans present as well as their location on the peptide chain. In mammalian cells, ER degradation-enhancing α-mannosidase-like proteins (EDEMs) acts as a mannose lectin and may be responsible for directing glycoproteins into the retrotranslocation and degradation pathway as overexpression of EDEMs has been show to accelerate the ERAD of misfolded proteins (Molinari et al., 2003; van Anken et al., 2003). EDEM bound substrates are then targeted to retrotranslocation elements such as derlin-2 or -3. In some cases EDEMs appear to compete for substrates with the calnexin/calreticulin cycle. The shift in favour of misfolded proteins to the ERAD pathway and binding by EDEMs is partly due to the fact that re-glucosylation by  17  glucosyltransferase and glucosidase II (necessary for calnexin/calreticulin rebinding) becomes considerably less efficient after mannose trimming (Grinna and Robbins, 1980; Sousa et al., 1992). Thus, due to its slow acting affects, EDEM may act as a timer to prevent proteins from being permanently cycled through re-glucosylation and folding (Fagioli and Sitia, 2001; Helenius and Aebi, 2001). However, sensors of misfolded proteins may also play an important role in ERAD. It appears that UGGT might be capable of directly detecting terminally misfolded proteins and thus targeting them for ERAD (Vembar and Brodsky, 2008). Calnexin itself has also been proposed to play a key role in sequestering mannose trimmed glycoproteins that are ERAD bound (Liu et al., 1997). In addition the number of glycans present and where they are located on the protein can also affect entry or exit from the calnexin/calreticulin cycle (Hebert et al., 1997). Lastly, some substrates may act sequentially or synergistically with BiP and calnexin while others may depend solely on BiP if the calnexin/calreticulin system has been compromised (Zhang et al., 1997). Prolonged BiP binding of hydrophobic patches of CFTR have been shown to recruit a ubiquitin (Ub) ligase (E3) to the BiP:CFTR complex (Meacham et al., 2001) and thus promote the degradation of these proteins upon misfolding. In addition to enabling disulfide links to form in newly synthesizing proteins, protein disulfide isomerases (PDIs) participate in the ERAD of many substrates (Gillece et al., 1999). PDIs work in tandem with BiP (Molinari et al., 2002; Svedine et al., 2004) and associate with calnexin or calreticulin (Oliver et al., 1999).  18  Once misfolded proteins have been identified and bound they are then generally shuttled out of the ER through retrotranslocons. Considerable evidence has suggested that the sec61 translocation channel has a secondary role as the retrotranslocation channel (Vembar and Brodsky, 2008). Other evidence also suggests derlins as an alternative retrotranslocon (Lilley and Ploegh, 2004; Ye et al., 2004). In addition to acting as the retrotranslocon, human derlins, can interact with other degradation components such as ubiquitination and targeting machinery in something of an ERAD complex. Derlin-1 has been shown to assist in HCMV-catalysed turnover of MHCI (Lilley and Ploegh, 2004; Ye et al., 2004), export from the ER during infection with the SV40 virus (Schelhaas et al., 2007) as well as efflux of an ERAD substrate in ER derived microsomes (Wahlman et al., 2007). Interestingly, a general translocon has not yet been found and the substrate specificity exhibited by the various recognition factors described above has confounded efforts to isolate a common translocon for ERAD substrates (Vembar and Brodsky, 2008). Furthermore, large proteins may be broken down prior to retrotranslocation and cytoplasmic or integral membrane portions may be translocated through different pathways or only partially degraded through ERAD mechanisms.  1.9.2 Ubiquitination of misfolded glycoproteins for ERAD  Most ERAD substrates are poly-ubiquitinated then transported to the proteasome for degradation (Helenius and Aebi, 2004; Raasi and Wolf, 2007; Vembar and Brodsky, 2008). In general, ubiquitination requires the activation of Ub through E1 binding, conjugation of Ub to an E2 enzyme and ubiquitination of target proteins via a specific E3 19  ligase (Johnston and Madura, 2004). The mode by which the target protein is ubiquitinated depends on the class of E3 protein that specifically interacts with that protein in that location. Thus E3’s are numerous and they fall into two major classes: RING domain E3’s permit specific interaction with Ub-E2’s, while the HECT class of E3’s form a covalent bond with Ub via E2 donation. E3 ligases have been found in association complexes with ERAD recognition factors and chaperone molecules as well as translocons themselves (Raasi and Wolf, 2007; Wang et al., 2006). For example, recent evidence has shown that the E3 ligase RMA1 and E2 ligase Ubc6e coassociates with derlin-1 to facilitate ERAD of CFTR and the misfolded deletion truncated CFTR mutant DeltaF508 (Younger et al., 2006). Ligase complexes may also act sequentially as in the case of the CFTR Delta F508 mutant, where RMA1 acts on the mutated protein in the ER and CHIP (an E4) acts on it in the cytosol. Very few E3 ligases have been specifically identified that regulate ERAD of common neurotransmitter receptors. In some cases, the E3 ligases that are known to modulate neurotransmitter receptor expression have been identified as acting or affecting these receptors at synaptic sites such as protein associated with myc (PAM) (Pierre et al., 2008), or KEL-8/CUL3 (Schaefer and Rongo, 2006) for the AMPA receptor subunit GluA1 and mGluR5 (PAM only). However, it is not clear if intracellular pools may contribute to the synaptic accumulation of these receptors. There is correlative evidence of an association between the E3 ligase gene UBE3A (also known as E6 associated protein) and downregulation of GABAergic transmission possibly via the concurrent reduction in GABA A receptor β3 subunit associated with  20  Angelman’s syndrome (Dan and Boyd, 2003; DeLorey et al., 1998; Samaco et al., 2005; Schroer et al., 1998). However again, a mechanistic relationship has not yet been elucidated. Thus, specific E3 ligases that mediate ERAD ubiquitination of many of the neurotransmitter receptors have yet to be identified. Given that there are potentially hundreds of E3s this is no great surprise (Ardley and Robinson, 2005).  1.9.3 Retrotranslocation and proteasomal degradation of misfolded glycoproteins  Many comprehensive reviews can be found that detail the complex machinery involved in the targeting of ubiquitinated proteins destined for the proteasome (Raasi and Wolf, 2007)., Briefly, upon the polyubiquitin tagging of misfolded proteins for ERAD, proteins must be extracted from the membrane. This is accomplished by the AAA+ ATPase p97 in mammals, the yeast homologue of Cdc48. Ufd1 and Npl4 adaptor proteins are conserved substrate targeting proteins that complex with p97 and Ub conjugated proteins. The p97 complex is maintained and recruited to the ER membrane by its interaction with Valosin-containing protein-interacting membrane protein (VIMP) (Ye et al., 2004). However, it is unclear how the ERAD substrates are recognized by the p97 complex. Some researchers have suggested that p97 may embed into the retrotranslocon to pull substrates into the cytoplasm via in an ATP dependent extraction, possibly through recognition of the polyubiquitin moieties (Flierman et al., 2003). Cdc48 has been shown to associate with the proteasome cap, however distinct Cdc48 proteasome-interacting factors may also bind with both the proteasome and the ubiquitinated target substrates. Upon polyubiquitination, a substrate is targeted to the 26S proteasome for degradation. The proteasome is made up of two 19S caps, which de21  ubiquitinate proteins and a 20S catalytic core, which contain multiple enzymes with trypsin, chymotrypsin-like and post-glutamylypeptide hydrolyzing activity to reduce the target protein into short oligopeptides (Vembar and Brodsky, 2008).  1.10 Degradation mechanisms of GABA receptors The majority of GABA-A receptors synthesized in heterologous cells are subject to polyubiquitination and ERAD (Bedford et al., 2001; Connolly et al., 1996; Saliba et al., 2007; Saliba et al., 2008; Figure 1.4). Poly-ubiquitination of newly synthesized proteins is a requirement for direction of misfolded proteins to the proteasome (Hoppe, 2005; Johnston and Madura, 2004; Koegl et al., 1999; Kohlmann et al., 2008; Nakatsukasa et al., 2008; Richly et al., 2005) whereas mono-ubiquitination or poly-ubiquitination of less than four Ub monomers directs target proteins from the cell surface to lysosomes for degradation (Chau et al., 1989; Johnston and Madura, 2004). Regulatory elements, such as Plic-1 (protein linking integrin-associated protein to cytoskeleton-1) that bind the Ub associated domains of GABA-A receptor α1-3, α6, and β1-3 subunits influence the receptor’s proteasomal degradation at the ER level (Bedford et al., 2001). Plic-1 coexpression with GABA-A receptors in HEK293 cells or over-expression in neurons has been shown to influence the receptors surface accumulation (Bedford et al., 2001; Saliba et al., 2008). Recently, Plic-1 has been shown to mediate this affect by increasing the stability of polyubiquitinated β3 subunits in the ER (Saliba et al., 2008). The authors propose that Plic-1 does not alter the rate of surface internalization, but under the right  22  Figure 1.4. Schematic diagram of the GABA-A receptor trafficking and degradation pathways. GABA GABA  Dynamin  Exocytosis  Endocytosis  Secretory vesicle  Clathrin-coated vesicle  Recycling Golgi Endosomal system  ERAD ERAD Proteasome degradation Endoplasmic Reticulum 23  Lysosomal degradation  circumstances, may protect newly synthesized GABA-A receptors from ERAD and thus influence the proportion of steady-state receptor accumulation. Similarly, it has been shown that activity dependent changes in GABA-A receptor number at the synapse may be mediated by ubiquitination and proteasomal degradation of newly synthesized GABARs in the ER (Saliba et al., 2007). Chronic blockade of synaptic activity with the voltage-gated sodium channel blocker tetrodotoxin (TTX) or glutamate receptor antagonists 6-cyanonitroquinoxaline-2,3-dione plus D-2-amino-5phosphonopentanoate (CNQX/D-AP5) in neuronal cultures resulted in increased ubiquitination of the GABA-A receptor and decreased surface stability. Consistent with this, picrotoxin treatment (a GABA-A receptor antagonist used to increase neuronal activity) stabilized GABA-A receptors at the surface and resulted in less GABA-A receptor ubiquitination. Interestingly, ERAD mechanisms appear to be mediating this affect. GABA-A receptors could still be degraded more rapidly than controls in the presence of TTX and the ER to Golgi transport inhibitor brefeldin A. Surface GABA-A receptors have been shown to undergo constitutive endocytosis in neurons and are degraded principally by the lysosomal pathway or recycled back to the surface (Kittler et al., 2004). Though surface GABA-A receptors can be rapidly endocytosed, under constitutive conditions, the fate of most internalized receptors is to be reinserted into the plasma membrane rather than be degraded. Binding of GABA-A receptors by huntingtin associated protein 1 is responsible for the direction of GABA-A receptors into a recycling pathway and overexpression of this protein has been shown to increase both the surface and synaptic accumulation of GABA-A receptors. Recently, the ubiquitination of seven lysine residues in the 2 intracellular loop between residues 325  24  and 335 has been shown to mediate the removal of GABA-A receptors from synaptic clusters and target them for lysosomal degradation in neurons (Arancibia-Carcamo et al., 2009). The importance of these residues in ubiquitination and targeting of receptors to lysosomes could be observed under both constitutive conditions as well as under conditions mimicking that of stroke using the oxygen glucose deprivation model. Blocking lysosomal degradation via the mutation of the lysine residues of the 2 subunit stabilized the GABA-A receptors in dendritic clusters after ischemic insult.  1.11 GABA-A receptor interactors and cell surface trafficking Using the yeast two-hybrid method, several interacting proteins have been identified in the trafficking of GABA-A receptors to the synapse (Tretter and Moss, 2008); Figure 1.5). GABA receptor associated protein (GABARAP) binds the interacellular loop of the 2 subunit and is thought to be important in the intracellular trafficking (Leil et al., 2004) of GABA-A receptors (Kittler et al., 2001; Kneussel, 2002; Tretter et al., 2008) through its interactions with the vesicular transport protein Nethylmaleimide-sensitive factor (NSF; Wang et al., 1999), tubulin (Wang and Olsen, 2000) and glutamate receptor interacting protein (GRIP1) (Marsden et al., 2007). In addition, scaffolding proteins, such as gephyrin, may be involved in the clustering of GABA-A receptors to the synapse (Essrich et al., 1998; Sassoe-Pognetto and Fritschy, 2000; Sassoe-Pognetto and Wassle, 1997) possibly through binding of α2 and more weakly with 2 (Tretter et al., 2008).  25  Figure 1.5. Proteins which interact with various subunits of GABA-A receptors.  Abbreviations: AP2, adaptor protein-2; BIG2, Brefeldin A-inhibited GDP/GTP exchange factor 2; CaMKII, Ca 2+/calmodulin dependent protein kinase II; GABARAP, GABA-A receptor-associated protein; GEC-1, GABA-A receptorassociated protein like 1; GODZ, Golgi-specific DHHC zinc finger protein; GRIF1, GABA-A receptor interacting factor-1; GRIP, Glutamate receptor interacting protein; HAP1, Huntingtin-associated protein 1; NSF, N-ethylmaleimide sensitive factor; PKA, Protein kinase A; PKC, Protein kinase C; Plic-1, Protein linking IAP and cytoskeleton 1; PP1, Protein Phosphatase 1; PP2B, Protein Phosphatase 2B; PRIP-1, Phospholipase C-related inactive protein type I; Src, sarcoma. (Modified from Chen and Olsen, 2007)  GRIP  Radixin PP2B Gephyrin  Plic-1  AP2  RACK1 CaMKII  GABARAP  p130 NSF GEC-1  GODZ  Src  PKC PKCβII  Akt  PKA  BIG2  AKAP150/79  gC1g-R PP1  PRIP-1 HAP1 GRIF-1  GRIP GABARAP  26  Alterations to the trafficking of receptors into and out of the PM may be a means to rapidly alter the strength of synaptic transmission (for a general review of endocytosis see Mukherjee et al., 1997; Wan et al., 1997). Endocytosis of GABA-A receptors has been shown to be dependent on a clathrin and dynamin dependent mechanism (Herring et al., 2003; Kittler et al., 2000; Figure 1.4). The adaptor protein, which mediates recruitment of target proteins for internalization, adaptor protein (AP2) complex binds both GABA-A receptor β and  subunits (Kittler et al., 2000). The regulated internalization of surface receptors appears to be dependent on an atypical AP2 binding motif in the intracellular loop of the β subunit (Kittler et al., 2005) and a YECL motif in 2 subunits (Kittler et al., 2008), but may also be regulated by the more common AP2 dileucine motif (Herring et al., 2003). Phosphorylation/dephosphorylation of the GABA-A receptor has been shown to occur at several sites within the β and  subunits and constitutes an important means by which the cell may internalize, stabilize or insert the receptor at the synapse to modulate synaptic inhibition. Several kinases that have been found to phosphorylate GABA-A receptors include the protein kinase A (PKA) (McDonald et al., 1998; Moss et al., 1992b; Porter et al., 1990), protein kinase C (PKC) (Krishek et al., 1994; Moss et al., 1992a), protein kinase B (PKB/Akt) (Wang et al., 2003) as well as Ca2+/calmodulin dependent kinase II (CaMK-II) (Houston et al., 2009), and the tyrosine kinase Src (Moss et al., 1995; Valenzuela et al., 1995). Phosphorylation can either potentiate or depress GABAA receptor function (Brandon et al., 2002; Kapur and Macdonald, 1996; Tretter and Moss, 2008). For example, phosphorylation of β3 at S408 and S409 enhances receptor function by blocking the atypical AP2 binding motif (Kittler et al., 2005; Terunuma et al.,  27  2008). In contrast β1 phosphorylation by PKA at S409 has been shown to reduce GABA-A receptor function (Brandon et al., 2003). Indeed alterations to GABA-A receptor surface number have been found to mediate changes in inhibitory transmission. For example, insulin stimulation causes a rapid insertion of GABA-A receptors into the plasma membrane (PM) via β2 phosphorylation (Wan et al., 1997; Wang et al., 2003). Increases in synaptic inhibition through receptor insertion, also occurs in response to NMDA receptor mediated long-term depression (LTD) through the involvement of CaMKII (Marsden et al., 2007). In contrast, a reduction in surface number may be induced by brain derived neurotrophic factor (BDNF Brunig et al., 2001) and is dependent on dynamin mechanisms (Hewitt and Bains, 2006). Internalization of GABA-A receptors has also been suggested to lead to overexcitation in the brain. Recent studies have shown that induction of status epilepticus (SE), which is an acute type of epileptiform activity characterized by prolonged and severe seizures, is associated with a rapid internalization of GABA-A receptors (Goodkin et al., 2005; Naylor et al., 2005). This rapid internalization may be mediated by the dephosphorylation of GABA-A receptor β3 subunits in the hippocampus (Terunuma et al., 2008).  1.12 Rationale, hypothesis and objectives Epilepsy is a debilitating disease affecting a significant proportion of the population. It is a complex disorder that may be induced due to trauma or manifest at any stage in development and thus, also has a complex etiology. Idiopathic epilepsies have been estimated to have a high genetic component, although most of the defective genes 28  remain unknown. However, some of the genes that have been linked to epilepsy affect the GABAergic system. This is unsurprising given that the inhibitory transmission has long been a common drug target to manage epilepsy. It remains, however to definitively link GABAergic defects with specific types of epilepsies. Identification of mutations in GABA-A receptor subunits associated with various forms of idiopathic generalized epilepsy have been rare, probably because these receptors are so critical to normal brain function. Previously, Cossette et al. studied the effects of a point mutation that was identified in a large French Canadian family that had heritable juvenile myoclonic epilepsy (Cossette et al., 2002). The mutation in the α1 subunit of the GABA-A receptor, α1(A322D) resulted in a substantial reduction in GABA currents recorded in HEK293 cells and western blotting suggested no alteration to the total expression levels observed for the mutant containing receptor. Thus, I hypothesized that the primary defect caused by the A322D point mutation was a reduction in the cell surface expression and trafficking of the GABA-A receptor. My objectives were to determine whether the A322D mutation 1) causes an intracellular trafficking alteration during secretion of the receptor through the ER, golgi to the cell surface and/ or 2) causes an instability of the mutant containing receptor at the cell surface and, lastly, 3) to elucidate the mechanism underlying any observed alterations that related to the epileptic phenotype.  29  CHAPTER 2 EXPERIMENTAL METHODS  2.1  Cell culture and plasmids Human embryonic kidney (HEK) 293 cells were cultured in culture media  consisting of Dulbecco’s modified eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Sigma) at 37 C, in a 5% CO2 humidified incubator. Cells were split with 0.5 % trypsin DMEM (Sigma) when cultures reached 90% confluence for continued culturing. Dishes were typically split one into three to a maximum of five dishes and considered the next generation. Cultures were generally used in experiments between 12 and 20 generations. Rat GABA-A receptor subunit cDNAs, pcDNA 3-β2, -2 were provide by Dr. Gholamreza Ahmadian and 2-GFP was a gift from Dr. H. B. Niznik (Clarke Institute of Psychiatry, Toronto, ON). The wild type FLAG epitope tagged pcDNA3-α1 construct was provided by Dr. J. H. Steinbach (Washington University School of Medicine, St Louis, MO) and was used as the template to generate the α1A322D mutant subunit using site directed mutagenesis by Cossette and co-workers (Cossette et al., 2002). Dynamin 1 wild type and K44E dominant negative constructs were provided by Dr. R.B. Vallee (University of Massachusetts, Shrewsbury, MA) (Herskovits et al., 1993). Caveolin-1 N terminal tagged GFP (pEGFP) construct was donated by Dr. A Helenius (Swiss Federal Institute of Technology Zürich, Zürich, Switzerland) (Pelkmans et al., 2001) and the green fluorescent protein (pEGFP) expression construct was purchased from Clonetech (Invitrogen). For analysis of the subunit dependence of the caveolin-1 interaction in 30  HEK293 cells, I used the pcDNA3 caveolin-1 myc tagged plasmid made by Dr. Bharat Joshi courtesy of Dr. Robert Nabi (University of British Columbia).  2.2 Transient transfections of HEK293 cells The day before transfection, cells were seeded evenly, on poly-D-lysine (PDL) coated or uncoated dishes at 2.5 or 3.5 x 106 cells / 10 cm dish, respectively. The cells were grown to 70% confluence and transiently transfected using Lipofectamine 2000 (Invitrogen) according to the manufactures’ protocols with some modification. One h prior to transfection, the normal culturing medium was replaced with 10 ml of fresh cold DMEM containing 5% FBS and incubated at 37 C as before. Transfection solutions were prepared as follows: 0.75 ml cold Opti-MEM (Invitrogen) was mixed with 30 µl Lipofectamine reagent per dish and incubated at room temperature (RT) for 15 min. DNA (10 – 12 µg) was mixed with 0.75 ml cold Opti-MEM per dish, combined with the Lipofectamine solution (total volume 1.5 ml / dish), mixed gently, and incubated for a further 15 min prior to adding to each plate. Unless otherwise stated, cells were transfected in the same media for 18 - 20 h at 37 °C with wild type rat FLAG epitope tagged α1 or α1(A322D) plasmid. In many experiments the α1 subunit (wild type or mutant) was co-transfected with rat β2, γ2 or γ2GFP plasmids in a 4:4:2 µg ratio. Single subunits were transfected at 4 µg / dish and two subunit combinations were transfected in a 4:4 µg ratio. In addition, some experiments also were co-transfected with 1.5 to 3 µg GFP to determine the health of the cells and transfection efficiency or as a control vector. Dynamin 1 wild type and K44E dominant negative constructs and Cav1-N terminal GFP plasmid were co-transfected with the 31  GABA-A receptor subunit plasmids at 1.5 to 3 µg / dish. Within each experiment, equal amounts of DNA were present in each condition and were of the same type of plasmid vector. This was done to avoid artifacts resulting from differences in the quantity of DNA present or rate of transcription of different vectors. In most experiments, transfection efficiency was assessed using GFP expression in at least one dish per experiment. This was typically 85-90% of total cells as assessed by visual inspection.  2.3 Cell ELISA assay for surface receptor expression levels HEK293 cells were transfected with wild type or mutant α1-FLAG constructs in a 2:1 ratio with β2 constructs (4 µg to 2 µg, respectively), to limit the number of receptors reaching the surface, plus GFP as described above. After 18-20 hrs incubation, transfected cells were rinsed with culture media, split as before with trypsin, equally plated onto PDL coated 12-well plates (approx 2–3 x 105 cells / ml / well) and incubated overnight at 37 °C in DMEM with 5% FBS. Cell ELISA’s were performed at RT, unless otherwise stated and all washes or rinses were done with Dulbecco’s phosphate buffered saline (D-PBS; Invitrogen; See Figure 2.1 for a schematic of the method). Transfected cells were rinsed, fixed for 10 min with 4 % paraformaldehyde and 4 % sucrose in PBS, and washed 3x 5 min on an orbital shaker at low speed to remove all fixation solution. Half the wells from each condition were permeabilized for 3 min with 0.1 % Triton X100 (Sigma) to determine total expression and 3x rinsed. Next, all the cells were blocked  32  Figure 2.1. Colorimetric cell ELISA assay used to quantify the surface expression of GABA-A receptors. Illustration of the steps involved in the immunodetection and quantification of surface versus total GABA-A receptors within the same cell.  Non-permeabilized  Permeabilized  HRP- 2 Antibody 1o Antibody  OPD substrate  Color development  Non-permeabilized values of optic density Permeabilized values of optic density  Relative surface expression 33  GABA-A receptor  for at least 1 h in 2 % goat serum (GS) and then incubated with either mouse anti-FLAG (Sigma) or polyclonal rabbit anti-α1 (Upstate) at a 1:500 dilution in 2 % GS for 1 h on an orbital shaker. The cells were washed 3x with D-PBS and then incubated with horseradish peroxidase (HRP) secondary antibodies (1:500; Amersham). Labelled cells were incubated with 1 ml of the chromagen substrate o-phenylenediamine (OPD; Sigma). The reaction was stopped according to wild type level color development with 200 µl of 3 N HCl and the chromagen product absorbances were read at 492 nm in a spectrophotometer. One control well from each condition was incubated with only secondary antibodies to determine background levels and used as the reference well for subsequent analysis. Surface levels of receptors, measured under non-permeabilized conditions, were determined as a ratio of the total receptor expression determined under permeabilized conditions.  2.4 Immunocytochemistry HEK293 cells were transfected with both known trafficking forms of the GABA-A receptor, α1β2γ2 or α1β2 (4:4:2 or 4:4 g ratio, respectively) and split into 12-well plates containing glass slides precoated with PDL. Cells were assayed the following day for either cell surface expression levels or co-localization with the early endosome marker Rab4, as described below. All wash and rinse steps were carried out at RT, with D-PBS on an orbital shaker. Coverslips were mounted with ProLong Gold anti-fade reagent (Invitrogen) overnight at RT. Images were collected on a Leica DMIRE2 deconvolution microscope using OpenLab software and exposure times were kept constant between samples. 34  2.4.1 Cell surface and intracellular expression immunocytochemistry  Cells were treated as described above and treated as illustrated in Figure 2.2. The next day, after a 1 h, 2 % goat serum blocking step, surface levels of α1 for wild type and mutant forms of the receptor were labeled with either anti-FLAG or anti-α1 diluted 1:1,000 or 1:500, respectively and detected with a 1 h incubation with anti-rabbit Cy3 secondary antibody (1:500; Jackson Immunologic Labs) in 2 % GS. These cells were then washed for 3x 10 min and then permeabilized with 0.1 % Triton X-100 for 5 min. After a 3x rinse, cells were incubated again with either anti-FLAG or anti-α1 primary antibody, washed 3x 10min, and the intracellular signal was detected with secondary antibodies conjugated to FITC (Jackson Immunologic Labs).  2.4.2 Colocalization of GABA-A receptors and early endosomes  Cells were washed and then permeabilized with 0.1 % Triton X-100 for 5 min to obtain the total signal for endosomes and GABA-A receptors to determine the extent of colocalization. After a 3x rinse, cells were blocked for 1 h with 2 % bovine serum albumin (BSA). Primary and secondary antibodies for each target were applied sequentially as follows: 1) Polyclonal rabbit anti-Rab4 1:250 in 2% BSA was incubated for 1 h at RT and rabbit secondary antibody Alexa Fluor 546 (Molecular Probes) at 1:1,000  35  Figure 2.2: Surface and intracellular immunostaining method for GABA-A receptor distribution. Illustration of the steps involved in immunodetection of surface and intracellular GABA-A receptors within the same cell.  Surface labeling  Surface with intracellular labeling  (Non-permeabilized) -  (Permeabilized)  Triton X-100  1  Antibody labeling  2 flurophore antibody labeling  1  Antibody labeling  36  2 flurophore antibody labeling  in 2 % GS was used to visual the signal; 2) Polyclonal rabbit anti-α1 (1:500) in 2 % GS was incubated overnight at 4 C and visualized with rabbit secondary antibody Alexfluor 488 (Molecular Probes) at 1:1,000 in 2 % GS. Because the primary antibody was the same, but used sequentially to look at surface versus intracellular populations of the receptor in the same cell, I permanently cross-linked the receptor with the primary and secondary antibodies to block any unbinding and subsequent competition for the next population of receptor when the cells were permeabilized.  2.5 Western blotting  2.5.1 HEK293 sample preparation for western blotting  Cells were washed 3x with cold D-PBS on ice and then lysed with 500 µl 0.5 % sodium dodecyl sulphate (SDS) in PBS containing a protease cocktail (10 µg / ml aprotinin, 10 µg leupeptin, and 1 mM phenylmethylsufonyl fluoride or 4- 2-aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF). Cell lysates were scraped off the dish with a rubber policeman and sonicated for 10 s, power level 3.5 with a Beckman Coulter Sonic Dismembrator 550. Lysates were then cleared of any unlysed cell and unsolubilized cell debris at 14k rpm for 20 min in a Beckman Coulter 22R refrigerated microcentrifuge. The supernatant was transferred to a new 1.5 ml tube and assayed for protein concentration with the BioRad DC protein assay kit, which is a detergent tolerant modification of the Lowry method. Samples were then diluted, and further denatured, with 2x Laemmli sample buffer (BioRad) and then boiled for 5-7 min at 95 C.  37  2.5.2 Denaturing polyacrylamide gel electrophoresis  Denaturing polyacrylamide gel electrophoresis (PAGE) was performed using discontinuous Tris-HCl gels made up of a 10 % acrylamide separating gel and a 4 % acrylamide stacking gel prepared with SDS to denature all proteins. A total of 30 - 60 µg lysate was subjected to SDS-PAGE in the mini-Protean III system (BioRad) and typically run at 120V for 1 h 20 min or until the dye front had reached the bottom of the gel. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) for 2 h at 120 V on ice or overnight at 42 V at 4 C.  2.5.3 Western blotting method  Membranes were removed from the transfer system, rinsed with distilled water and then tris buffered saline with Tween-20 (TBST) containing 20 mM Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.5. Membranes were blocked in 5 % skim milk / TBST for 20 min at RT and then transferred to the primary antibody solution. Depending on the optimal conditions for the primary antibody binding, membranes were incubated with the primary solution for 1 to 2 h at RT or overnight at 4 C (see section 2.5.4). Blots were rinsed 3x with distilled water and washed in TBST 2x for 10 min to remove trace primary antibody solution. Secondary antibody incubation was for 1 h at RT using goat HRPconjugated secondary antibodies diluted 1:2,000 – 5,000 in 1% skim milk TBST. Immunoreactive bands were visualized with the enhanced chemiluminescence detection reagent kit (Amersham). Images of immunoblots were obtained with a BioRad imager and Quantity One software (BioRad). ImageJ software (NIH) was used to quantify the  38  relative signal intensities from each band. All samples were analyzed relative to one another on the same membrane and within the same experiment.  2.5.4 Western blotting antibodies and primary antibody conditions.  The primary antibodies used for western blotting were diluted in 5 % skim milk with TBST, generally overnight at 4 C unless otherwise stated, and included the following: 1) Mouse monoclonal M2 anti-FLAG (Sigma; F3165) diluted 1:1,000 at RT for 1hr 2) Rabbit polyclonal anti-rat GABA-A receptor α1 subunit (Upstate; 06-868) diluted 1:500 3) Rabbit polyclonal anti-rat GABA-A receptor intracellular loop 2 subunit (raised by Dr Gholamreza Ahmadian) diluted 1:100 4) Mouse monoclonal anti-rat GABA-A receptor β2/3 subunit (Upstate; 05-474) diluted 1:500 or 1:1,000 5) Rabbit polyclonal anti-caveolin-1( BD Transduction Labs; 610060) diluted 1:500 (for HEK293 cells) 6) Rabbit polyclonal anti-GFP (Chemicon; AB3080) diluted 1:1,000 in Signal Enhancer Hikari (Nacalai USA; NU00102) 7) Rabbit polyclonal anti-calnexin (Stressgen; SPA-865F) diluted 1:1,000 in 1% skim milk TBST, at RT for 1hr 8) Mouse monoclonal anti-Ub (Chemicon; MAB1510) diluted 1:1,000 in TBST  39  Note that most of the diluted antibody solutions were preserved with 1 % sodium azide and stored at 4 C for multiple reuse (typically 6-15 blots, depending on the stability of the antibody).  2.6 Coimmunoprecipitation assays  2.6.1 Examination of transfected α1 binding with endogenous calnexin  For analysis of the proportion of calnexin associated with the wild type and mutant α1 subunits and various subunit combinations, HEK293 cells were lysed with modified radioimmunoprecipitation assay (RIPA) buffer A (150 mM NaCl, 50 mM HEPES, 1% NP-40, 1 mM EDTA) containing a protease inhibitor cocktail, as described earlier, on ice. Lysates were further homogenized with various gauge needles moving from largest to smallest (16 G, 22 G, 25 G) 6x each and spun down to remove unlysed cell debris for 20 min at 14k rpm, at 4 C. Protein assays were performed as described with the BioRad DC protein assay kit. To co-IP proteins associated with the α1 subunit, 10 µg of FLAG antibody was complexed to 40 µl 50% protein A sepharose bead slurry (Amersham) for 2 h at RT. Antibody bound beads were washed 3x with 1 ml D-PBS to remove unbound antibodies from the bead-antibody slurry. The lysates (0.5 mg per sample) were diluted to 1 ml with D-PBS and precleared with 30 µl protein A sepharose bead slurry. Precleared lysates were immunoprecipitated for 2 - 4 h, rotating at 4 °C with the prepared antibody-bead slurry. After a 5x wash with 1 ml D-PBS plus protease inhibitors, precipitated proteins were eluted from the beads with 75 µl 2x Laemmli buffer boiled for 5 – 7 min at 95 C. 40  SDS-PAGE, immunoblotting, and band intensity quantification was carried out as described earlier. Membranes were probed for the α1 subunits and then reprobed with a polyclonal rabbit calnexin antibody. I also probed for GFP as a further control for receptor assembly in HEK293 cells. I used TrueBlot rabbit HRP secondary antibodies (eBiosciences), which only detect light and heavy chain Ig’s that are still bound to one another and thus do not interfere with visualization of the immunoreactive bands after denaturation. After obtaining signal intensity values from all the bands, the proportion of calnexin signal intensity relative to α1 intensity in each sample was measured.  2.6.2 Coimmunoprecipitation of the GABA-A receptor α1 subunit:caveolin 1 protein complex in transfected HEK293 cells  HEK293 cells cotransfected with the wild type FLAG tagged GABA-A receptor α subunit, myc tagged caveolin 1, and GFP plasmids (4:3:2 µg) were lysed with 400 µl modified RIPA buffer A and protein assayed as described earlier. Immunoprecipitation of caveolin-1 was done largely as described earlier, but with a rabbit polyclonal anti-caveolin 1 (BD Transduction Labs) conjugated to 40 µl of beads at RT as it appeared to detect this caveolin construct with greater affinity than the Santa Cruz antibody. For immunoprecipitation of the GABA-A receptor α1 subunit, I used the FLAG tagged protein immunoprecipitation kit (Sigma) containing bead-conjugated mouse monoclonal FLAG M2 antibodies. Beads (50 µl / sample) were washed 3x with 0.75 ml of the supplied 1x wash buffer and then incubated for 2-4 hrs at 4 C with precleared 1 mg / ml diluted lysates. Immunoprecipitates and controls were washed 3-4x  41  with 1x wash buffer and then eluted from the antibody-bead complex with 50 µl [300 ng / µl] 6X FLAG peptide in 1x wash buffer after a 30 min competitive elution at 4 C and gentle shaking. Eluted proteins were heat denatured as before and western blotting was carried out as described earlier.  2.7 Metabolic pulse-chase assays Twenty to 24 h post-transfection with α1 or α1β2γ2 the transfection media was replaced with 5 ml fresh 5% FBS DMEM for 1-2 h. This and all subsequent incubations were carried out at 37 °C. Media was replaced again with 4 ml methionine free DMEM (Invitrogen) for a period of 30-45 min. Expressing proteins were radiolabeled with 100 µCi / ml 35S-methionine (PerkinElmer) for 30 min, washed 3x with D-PBS, and the medium was changed to chase medium containing 100 µg / ml cycloheximide (Sigma) in DMEM to block further protein synthesis for 0, 60, 120, 240 or 480 minutes. At the end of the chase times, cells were washed 3x with ice cold D-PBS and lysed in 650 µl PC lysis buffer (0.5% SDS, 1% NP-40, 5 mM EGTA, 5 mM EDTA in PBS with a protease inhibitor cocktail). GABA-A receptor α1 subunits were immunoprecipitated from radiolabelled lysates (1- 2 mg / ml) with anti-FLAG M2 agarose affinity gel (40 µl) and incubated rotating at 4 °C for 2 - 4 h. Immunoprecipitated proteins were eluted from the beads with 2x Laemelli Buffer and loaded on a 4 % stacking / 12% separating acrylamide midi-gel (Hoeffer). These gels were run at 4 °C overnight or until the dye front band had reached the bottom of the gel (generally run at 80 V for 2 hrs then 50 V for the remainder of the time). The gels were fixed in a solution of 10 % acetic acid, 40 % methanol in water for at least 20 min and then transferred to the detection enhancing reagent Amplify 42  (Amersham) for a further 20 min. Gels were dried on a gel dryer for 1.5 – 2 h at 70 °C and exposed to autoradiograph film (Amersham) at -80 °C for 1 – 2 days. Lastly, the bands of interest and background regions for each lane were excised from the gels and digested overnight in 200 µl 65 % perchloric acid and 400 µl hydrogen peroxide added to gel bands dropwise. Bands were digested for 3 h at 60 C and incubated overnight at RT. The next day scintillation cocktail (Amersham) was added to the dissolved samples, mixed on a vortexer for 30 s, and then left at RT for 1 h to remove air bubbles. The samples were then quantified for radioactivity in counts per minute (cpm). Background levels were subtracted from each excised band and the normalized cpm for each time point was averaged. The half life was calculated by linear regression to determine the y intercept and slope. This slope is the decay constant (k) in the relationship: At = A0e–kt . The data showed first order kinetics for all four samples and so the half lives were calculated using the single exponential relationship: t1/2 = ln2/k, where ln2 is equal to 0.693.  2.8 Treatment conditions  2.8.1 Degradation blockade assay conditions  Transfections were performed with the full receptor complex as described earlier, but shortened to a 6 h incubation time. The cells were washed and allowed to recover in fresh 10 % FBS DMEM for 2 h and then treated with or without (controls) water soluble lactacystin (2R)-2-(acetylamino)-3-[(carbonyl)sulfanyl]propanoic acid (BioMol International; 10 or 15 µM) or leupeptin N-(acetyl-L-leucyl-L-leucyl-L-argininal)  43  (Sigma; 25 or 50 µM) overnight at 37 C. Cells were processed for western blotting as described above.  2.8.2 Protein folding assay conditions  Transfection and pre-treatment conditions were similar to degradation assays. After the recovery from transfection incubation, cultures were treated with or without 0.25-0.5 % dimethylsulfoxide (DMSO) overnight at 37 C. Analysis for a temperature sensitive misfolded protein was determined by carrying out the transfections as before, but splitting sister cultures into either the 30 C low temperature or the normal control 37 C conditions. I used western blotting to determine whether DMSO or low temperature increased or decreased the rate of degradation relative to controls.  2.8.3 Dynamin and caveolin blockade conditions  Transfections were carried out with 1.5 to 3 µg of the dominant negative constructs K44E dynamin 1 or caveolin 1-N terminal GFP co-expressed with either wild type or A322D mutant GABA-A receptors. Controls for the each dominant negative construct, wild type dynamin 1 or GFP (for caveolin 1), were likewise co-expressed with either wild type or A322D mutant GABA-A receptors. Optimization of the amount of DNA transfected for each DN construct was based on the health of the cells expressing the mutant GABA-A receptor as these cells were the more sensitive to toxicity resulting from over-expression.  44  2.8.4 N-glycosylation blockade with 35S-methionine labeling Cells transfected with either the α1 or the α1(A322D) GABA-A receptor subunit were treated with tunicamycin to block N-glycosylation of these subunits and were assayed similarly as in the metabolic pulse-chase assay, but with some differences. Specifically, cells were pretreated with or without 10 µg / ml tunicamycin (Sigma) to block N-glycosylation in culture medium for 1 h and treated again with methionine free starvation media (methionine free DMEM; Invitrogen) for 30 min. Cells were then pulsed with 200 µCi / ml 35S-methionine in starvation media for 1-2 h, with or without tunicamycin, and the α1 subunits were anti-FLAG immunoprecpitated using 1 mg total protein. The remainder of the protocol was carried out as described for the pulse-chase assay and individual bands were visualized by exposure to autoradiographic film (Kodak) for 3 h at -80 C.  2.9 Surface biotinylation assays HEK293 cells were transfected as before to express the wild type or mutant receptors in the presence of wild type dynamin, control GFP, or the dominant negative constructs dynamin-1 K44E or Caveolin-1 N terminus GFP to assess the effects on the surface receptor number after endocytosis blockade. All cells were washed 2x with 5 ml and then once with 10 ml sterile cold PBS and incubated with 4 ml cold HEK293 extracellular solution (ECS in mM: 140 NaCl, 1.5 CaCl2, 1.3 KCl, 25 HEPES, 33 glucose (pH 7.4, 310-320 mOsm) for 30 min at 4 C. I added 1 ml of a 5 mg/ml EZ-Link SulfoNHS-LC-Biotin (Pierce Biotechnology) diluted in ECS solution to each sample, mixed  45  gently, and incubated again at 4 C to label surface proteins. Cultures were washed thoroughly on ice with 10 ml D-PBS, lysed with 600-750 µl biotin lysis buffer made of 0.5 % SDS in PBS with protease inhibitor cocktail. Samples were homogenized with syringes, spun down to remove cell debris and protein assayed as described before. Biotin labeled proteins were pulled down with 100 µl per sample prepared avidin agarose from egg white aqueous glycerol suspension (Sigma). Slurries were spun down briefly (15 s at 14K rpm), washed 3x with 0.5 % SDS PBS to remove the glycerol preservative, and resuspended in blocking buffer (10 mg/ml bovine albumin serum diluted in biotin lysis buffer) to give a 50 % v/v bead slurry. Lysates were diluted 0.5 - 1 mg / ml with blocking buffer and incubated at 4 C rotating with the avidin beads to allow binding to occur. Beads were pelleted and washed 1x with PBS, 3x with 0.5 M NaCl PBS, and 2x with PBS to remove trace salts. After the last spin, I added 70 – 75 µl 2x Lamelli sample buffer and eluted the bound proteins at 95 C for 7 min. Samples were processed for western blotting as described.  2.10 Amino acid sequence alignment Amino acid sequences for all rat GABA-A receptor α subunits were obtained from the protein database on NCBI (GABRA1: NP899155, GABARA2: NP001129251, GABRA3: NP058765, GABRA4: NP542154 , GABRA5: NP058991, GABRA6: NP068613) and exported for sequence alignment into the online version of CLUSTALW (provided by NIH).  46  2.11 Statistical analysis I used a two-tailed, paired or unpaired Student t-test as appropriate to assess statistical significance and set significance at P<0.05 for within sample analysis. Statistics were performed on raw optical density measurements excepting analysis of percent changes between wild type and mutant groups. Specifically, to assess statistical significance for percent changes after treatments or in the presences of dominant negative constructs between wild type and mutant GABA-A receptor types, I performed a twotailed, unpaired Student t-test significance and set significance at P<0.05. All quantifications are expressed as the mean ± standard error.  47  CHAPTER THREE RESULTS  3.1 Introduction Roughly 0.4 to 1% of the population suffers from various forms of idiopathic generalized epilepsy (Baumann et al., 2002; Connolly et al., 1996; Mehta and Ticku, 1999). However, many of these forms of epilepsy show significant inheritability of this disorder. While a family link can be observed at times, complex transmission and variability of symptoms confounds classification of specific epilepsy phenotypes to particular genes. Our lab had studied the effects of a point mutation that was identified for the first time in the α1 subunit of the GABA-A receptor isolated from analysis of a large French Canadian family that had heritable juvenile myoclonic epilepsy (Cossette et al., 2002). Functional characterization of the α1(A322D) mutation showed a drastic reduction in GABA currents recorded in HEK293 cells. This reduction in GABA responsiveness could be due to one or more dysfunctions caused by the mutation, including impairment in α1subunit synthesis, impaired receptor assembly, altered trafficking, cell surface instability, enhanced degradation or an alteration to channel function in the mutant containing receptors. Preliminary western blotting results from the laboratory had indicated no change to the receptor number. Therefore, I hypothesized that the primary defect caused by the α1(A322D) mutation was a reduction in cell surface expression of the GABA-A receptor due to reduced trafficking in the secretory pathway and/or enhanced instability of the receptor at the cell surface.  48  In the course of this study, other groups also reported the effects of this mutation in GABA receptors expressed in HEK293 cells (Fisher, 2004; Gallagher et al., 2007; Gallagher et al., 2005; Gallagher et al., 2004; Krampfl et al., 2005), and these studies will be considered in relation to my own work in the discussion.  3.2 Results  3.2.1 The effects of the 1(A322D) mutation on the cell surface expression of GABA-A receptors expressed in HEK293 cells.  To address cellular trafficking or stability changes resulting from the point mutation, I first examined whether the mutation changed the surface expression of recombinant rat GABA-A receptors. I compared the surface and intracellular levels of the wild type and mutant assemblies, using the N-terminal FLAG epitope tagged 1 or N-terminal FLAG epitope tagged 1(A322D) constructs expressed in HEK293 cells (Cossette et al., 2002) and performed double label immunocytochemistry (see Figure 2.2 for method used). For these experiments, I co-expressed the β2 subunit so as to be able to form a trafficking competent receptor that could be expressed on the membrane surface. In all cells imaged, the wild type (i.e., α1β2) combination was readily detected on the membrane surface as well as intracellularly, as determined following permeabilization (Figure 3.1). In contrast, the mutant assembly (i.e., α1(A322D)β2) showed pronounced intracellular staining, but little or no surface labeling when exposure  49  Figure 3.1. The α1(A322D) mutation causes a marked reduction in the cell surface expression of GABA-A receptors observed by immunocytochemistry.  An imaging comparison of the distribution of wild type and mutant α1 subunits on the surface (non-permeabilized) and intracellularly (permeabilized) when coexpressed with β2 subunits in HEK293 cells. Images were taken at equal exposure times, calibrated to the wild type signal within the mid-range of signal intensity. Merged images are composites of equal signal contributions from each image. These are representative images of experiments that were performed on numerous occasions, using either the α1β2 or α1β22 combinations in HEK293 cells.  50  Figure 3.1. The α1(A322D) mutation causes a marked reduction in the cell surface expression of GABA-A receptors observed by immunocytochemistry.  Non -permeabilized  Permeabilized  Wild type  Mutant  51  Merged  times were optimized for wild type surface expression. Identical results were also observed when I additionally co-expressed the γ subunit (not illustrated). To quantify the reduction in surface expression of the mutant GABA-A assembly, I used a colorimetric cell ELISA approach (Figure 2.1). Cell surface expression of wild type (12) receptor averaged 44 ± 8 % of the total cell expression. In contrast, cell surface expression of the mutant 1(A322D)2 receptor was only 13 ± 3 % of total cell expression. This difference was statistically significant (Figure 3.2A, N=3; P < 0.05). Together, these results show that a smaller proportion of mutant GABA receptors are present on the cell surface compared to wild type GABA receptors.  3.2.2 The effects of the 1(A322D) mutation on total expression of GABA-A receptors expressed in HEK293 cells.  Surprisingly, given previous work (Cossette et al., 2002), the cell ELISA also revealed a significant decrease in the total expression of the mutant assembly (to 44 ± 1% of control; N=3; P < 0.05; Figure 3.2B). To more readily quantify the effects of the mutation on total GABA-A receptor expression, I performed western blots. For these experiments I initially compared the effects of the mutation on the α1 subunit alone. I found that the A322D mutation caused a reduction in the expression of the single α1 subunit (73 ± 1% of control; N=5, P<0.01; Figure 3.3). I next investigated a trafficking-competent receptor. For these experiments I also expressed the γ2 subunit, along with the β2 subunit, since the α1β2γ2 assembly is the most common trafficking competent receptor subunit combination 52  Figure 3.2. The A332D mutation results in a reduction in both surface expression and total expression of GABA-A receptors.  A. Cell ELISA quantification of GABA-A receptor surface expression relative to total expression in mutant (1(A322D)2) and wild type (12) transfected HEK293 cells (N=3). B. Cell ELISA analysis for total 1(A322D)2 expressed relative to α1β2 (N=3) shows reductions in total mutant containing receptors. * P<0.05.  53  Figure 3.2. The A332D mutation results in a reduction in both surface expression and total expression of GABA-A receptors.  A 60  *  50 40 30 20 10 0  Wild type  Mutant  B 60  * 40 20 0  Mutant 54  Figure 3.3. The A322D mutation leads to reduced total expression of the α1 subunit.  Western blot analysis of α1 expression in HEK293 cells transfected with the wild type (α1) or the mutated A322D form (α1′) alone (N=5). A representative blot is shown above the corresponding histogram. Blot intensity values have been normalized relative to wild type levels. ** P<0.01.  55  Figure 3.3. The A322D mutation leads to reduced total expression of the α1 subunit.  1  1.2  m m  1.0 0.8  **  0.6 0.4 0.2 0  Wild type  56  Mutant  (Connolly et al., 1996). As a control, I also performed a co-IP with anti-FLAG to pull down α1 wild type and mutant subunits and immunoblotted to confirm incorporation of the 2-GFP subunit into the full receptor complex (Figure 3.4A). I then performed westerns using the α1β2γ2 assembly (which is referred to hereafter as the receptor). There was a pronounced reduction in the expression of the mutant receptor (46 ± 7% of control; N=6, P<0.01; Figure 3.4B). Interestingly, the mutation causes a more dramatic effect on the expression of the receptor than on the isolated α1 subunit (P<0.01), an effect that was comparable to that observed on the α1β2 assembly in the cell ELISA assays.  3.2.2 The A322D mutation causes greater cell stress upon expression in HEK293 cells and neurons.  Throughout this work, it was apparent that the HEK293 cells expressing the mutant form of the receptor were much more labile. Many experiments had required optimization of the treatment conditions to ensure that cultures were not skewed by greater cell death in mutant transfected cells. Cell cultures were regularly examined posttransfection and throughout treatment conditions using membrane impermanent cell dye reagents such as propidium iodide to determine cell death. However, all attempts to transfect neuronal cultures using lipofectamine or nucleofection under a variety of conditions with the mutant receptor laid waste to all the apparently transfected cells (GFP co-expressing), while sister cultures expressing wild type GABA-A receptors were easily and robustly expressed. This led me to wonder whether the mutant receptor causes additional ER stress such that the unfolded protein response (UPR) was engaged.  57  Figure 3.4. The A322D mutation results in a dramatic decrease in the total expression of the GABA-A receptor.  A. Immunoblotting (IB) for the cotransfected 2-GFP subunit demonstrates that immunoprecipitation (IP) of the FLAG tagged α1 subunit with anti-FLAG (wild type or mutant types) co-IPs the 2-GFP. This demonstrates that the γ2 subunit has been successfully incorporated into the full receptor complex. B. Western blotting analysis of α1 expression in HEK293 cells transfected with the wild type (α1β2γ2) or the mutated A322D form (α1′β2γ2) (N=6). A representative blot is shown above the corresponding histogram. Blot intensity values have been normalized relative to wild type levels. ** P<0.01.  58  Figure 3.4. The A322D mutation results in a dramatic decrease in the total expression of the GABA-A receptor.  A IP: Flag IB: 1 IB: GFP  B 1  20  1.2 1.0 0.8 0.6  **  0.4 0.2 0  Wild type  59  Mutant  I therefore probed western blots of mutant and wild type receptor lysates using an antibody against BiP/Grp78, a critical ER chaperone that becomes upregulated in response to ER stress and is an early indicator of activation of the UPR. I tested an antibody against human BiP/Grp78 (Stressgen) under a variety of blotting conditions, but was unable to produce blots that had a sufficient signal-to-noise ratio for quantification. However, while quantitatively inconclusive because of a low n value, visual inspection of two blots showed no obvious change in BiP expression levels in wild type or mutant expressing HEK293 cells. This suggested that the UPR may not be engaged under these circumstances.  3.2.4 The A322D mutation causes enhanced association with the ER chaperone calnexin.  Even if the UPR was not activated, enhanced misfolding of the receptor could be putting undue pressure on the cell. Continued synthesis of the mutant receptor over time may have the cumulative effect of exhausting the cell as it also has to continually degrade it. I reasoned that if the mutation caused a misfolding of the subunit then the mutant subunit and/or the mutant receptor could be targeted for ER associated-degradation (ERAD). One way to test for this is to assess its ability to bind to the ER chaperone calnexin, which is known to bind GABA-A receptor subunits in the ER via their Nterminal glycosylation moieties (Connolly et al., 1996). Prolonged association with calnexin (and other ER chaperones) retains misfolded proteins in the ER and helps tag them for subsequent degradation (Helenius and Aebi, 2004; Moremen and Molinari,  60  2006; Parodi, 2000). I investigated this possibility using co-IP in HEK293 cells transiently expressing the wild type or mutant α1, along with or without β2, γ2 or β2γ2 constructs. Consistent with an increase in targeting for ERAD, I found an approximately threefold increase in association of the mutant receptor (1.48 ± 0.15, N=4) compared with the wild type receptor (0.48 ± 0.04; N=4, P<0.01; Figure 3.5) with calnexin. However, the total amount of endogenous calnexin remained the same in HEK293 cells transfected with wild type or mutant receptor (N=4; Figure 3.6). A similar difference (ranging from 2.2 to 2.5 fold) in calnexin to α1 ratio was observed irrespective of whether the α1, α1β2 or α1γ2 combinations were examined. The single wild type α1 subunit association with calnexin was 0.61 ± 0.08 while the single mutant α1 subunit association with calnexin was 1.50 ± 0.16 (N=4, P<0.05; Figure 3.7). When cotransfected with the β2 subunit, the wild type association was 0.76 ± 0.10 while the mutant association with calnexin was 1.68 ± 0.17 (N=4, P<0.01; Figure 3.8). When cotransfected with the 2 subunit, the wild type association was 0.72 ± 0.09 while the mutant association with calnexin was 1.67 ± 0.52 (N=4, P<0.05; Figure 3.9). These results suggest that the mutation causes a protein misfolding which leads to calnexin-dependent retention and subsequent targeting to ERAD and this is independent of the subunit combination expressed.  61  Figure 3.5. The A322D mutation increases the co-association of GABA-A receptor subunits with the endoplasmic reticulum chaperone calnexin.  A plot of the calnexin : α1 subunit ratio for wild type and mutant subunits (N=4). FLAGtagged α1 subunits were immunoprecipitated (IP) with FLAG antibodies. The representative immunoblots (IB) above the histogram show the level of endogenous calnexin (cal) bound to α1 subunits for non-transfected cells (Cont) and cells transfected with wild type and mutant forms of the α1β2γ2 receptor. ** P <0.01.  62  Figure 3.5. The A322D mutation increases the co-association of GABAA receptor subunits with the endoplasmic reticulum chaperone calnexin.  IP: Flag IB: 1 IB: Cal  2.5 2.0  ** 1.5 1.0 0.5 0  Wild type  63  Mutant  Figure 3.6. Expression of the mutant form of the GABA-A receptor does not alter the total amount of calnexin.  Western blotting shows that the total levels of calnexin (Cal) were similar in HEK293 cells expressing wild type α1β22 and mutant α1′β2γ2 (N=4). A representative blot is shown above the corresponding histogram and includes a blot of non-transfected control (Cont) cells. Total calnexin intensity units are expressed as arbitrary values.  64  Figure 3.6. Expression of the mutant form of the GABA-A receptor does not alter the total amount of calnexin.  IB: Cal  6000  4000  2000  0  Wild type  65  Mutant  Figure 3.7. The A322D mutation increases the co-association of the α1 subunit with calnexin.  A plot of the calnexin : α1 subunit ratio for wild type (α1) and mutant (α1′) subunits expressed in HEK293 cells alone (N=4). FLAG-tagged α1 subunits were immunoprecipitated (IP) with FLAG antibodies. Representative immunoblots (IB) above the histogram show the relative levels of endogenous calnexin (Cal) that were found to be co-associated with the α1 subunit types. * P <0.05.  66  Figure 3.7. The A322D mutation increases the co-association of the α1 subunit with calnexin.  IP: Flag IB: 1 IB: Cal  2.5 2.0  *  1.5 1.0  0.5 0  1  67  1 1  Figure 3.8. The A322D mutation similarly increases the co-association of the α1β2 receptor with calnexin.  A plot of the calnexin : α1 subunit ratio for wild type (α1β2) and mutant (α1′β2) GABAA assembly expressed in HEK293 cells (N=4). FLAG-tagged α1 subunits were immunoprecipitated (IP) with FLAG antibodies. Representative immunoblots (IB) above show the relative levels of endogenous calnexin (Cal) that were found to be co-associated with the α1 subunit types when co-expressed with β2 subunits. ** P <0.01.  68  Figure 3.8. The A322D mutation similarly increases the co-association of the α1β2 receptor with calnexin.  IP: Flag IB: 1 IB: Cal  2.5  **  2.0 1.5 1.0 0.5 0  1  1 1  + 2  69  Figure 3.9. The A322D mutation likewise increases the co-association of the α12 combination with calnexin.  A plot of the calnexin : α1 subunit ratio for wild type (α12) and mutant (α1′2) combinations of GABA-A receptor subunits co-expressed in HEK293 cells (N=3). FLAG-tagged α1 subunits were immunoprecipitated (IP) with FLAG antibodies. Representative immunoblots (IB) above the histogram show the relative levels of endogenous calnexin (Cal) that were found to be co-associated with the α1 subunit types when co-expressed with 2 subunits. ** P <0.01.  70  Figure 3.9. The A322D mutation likewise increases the co-association of the α12 combination with calnexin.  IP: Flag IB: 1 IB: Cal  2.5  *  2.0 1.5 1.0 0.5 0  1  1 1  + 2  71  3.2.5 Lowering the incubation temperature does not affect the expression of mutant α1 containing GABA-A receptors.  I next sought to examine whether the enhanced degradation seen with the α1(A322D) mutant could be rescued with treatments that lower the Gibbs free energy (G) of the protein folding reaction (i.e., less energy is required to correctly fold the protein). Thus, lowering the G for the reaction effectively enhances the correct folding rates of proteins. Since many mutant proteins have been found to be temperature sensitive, I examined whether lowering the incubation temperature to 30 C could rescue the α1(A322D) trafficking competent receptor (Figure 3.10). There was a reduction in the total expression of both wild type and mutant α1β2γ2 receptors at 30 C relative to 37 C (Figure 3.10A). In arbitrary optical density units (OD) wild type and mutant α1 total expression was 4,983 ± 90 and 1,779 ± 409 at 30 C relative to 7,100 ± 542 and 3237 ± 785 at 37 C. In terms of the effect of temperature on the assembly of the mutated receptor there was no apparent difference. The mutant to wild type receptor ratio at 37 C (46 ± 10%; N=3) and the mutant to wild type receptor ratio at 30 C (36 ± 8%; N=3) were not significantly different (Figure 3.10B). Thus, it appeared that reducing the temperature did not preferentially affect the expression of the mutant receptor relative to the expression of the wild type receptor.  72  Figure 3.10. Low temperature incubation does not alter the ratio of wild type to mutant GABA-A receptor.  Wild type (Wt) and Mutant (Mut) GABA-A receptors (α1β2γ2) transfected and incubated at 30 C were compared to that of control conditions at 37 C. A. Incubation at 30 C resulted in a decrease in expression of both wild type and mutant α1 containing receptors. The graph plots the levels of α1 expressed normalized to wild type cells at 37 C incubation (N=3). B. Plots the ratio of the level of expression of the mutant receptor versus the wild type receptor at each temperature. * P<0.05.  73  Figure 3.10. Low temperature incubation does not alter the ratio of wild type to mutant GABA-A receptor.  A  37C  30C  Mut  Wt  Wt  Mut  1 1.4 1.2 1.0 0.8  *  0.6 0.4  *  0.2 0  Mut  Wt  Wt  37C  B  Mut 30C  NS  60  *  50  *  40 30 20 10 0  37C 74  30C  3.2.6 A chemical chaperone partially rescues the expression level of mutant containing GABA-A receptors.  Another way to assess whether the A322D containing receptor is more likely to misfold is to use chemical chaperones. Mechanistically, these chaperones are membrane permeant and work typically by creating new interactions with the nascent protein chain which then provide opportunities for the “correct” folding interaction to occur without exerting excess force (Papp and Csermely, 2006). In pilot experiments, I tested a range of concentrations of the chemical chaperone, dimethylsulfoxide (DMSO) between 0.13% and 4.0% (v/v). Though wild type expressing cells were relatively untroubled, I found that 1% DMSO or higher caused toxicity in mutant expressing HEK293 cells. In addition, I found that shortening the transfection time and adding a 1-2 hour recovery step helped keep the cells viable. One effect of this alteration in transfection time, however, was a drop in the total overall levels of both receptor types expressed in these cells. This also resulted in increased variability of untreated mutant receptor expression levels. I increased the number of replications for all conditions to compensate and pooled data for treatments of 0.5% and 0.25% DMSO in wild type and mutant expressing HEK293 cells to determine whether the degradation related to the A322D point mutation could be protected by a chemical chaperone (Figure 3.11). With DMSO treatment, I found there was no significant effect on wild type receptor expression levels (measured as arbitrary optical density units) at these concentrations (10,770 ± 451) relative to wild type untreated controls (11,106 ± 567; N=9 treated, N=5 untreated, unpaired t-test P=0.67). However, there was a trend towards increased levels of mutant receptor in  75  Figure 3.11. The protein folding chemical chaperone DMSO partially increased the expression levels of mutant containing GABA-A receptors.  A. Western blots show that treatment with the chemical chaperone DMSO (0.25-0.5%) partially increased the levels of the mutant (Mut) α1β22 containing receptors in HEK293 cells. The graph plots the levels of total α1 subunits expressed in arbitrary units with or without DMSO treatment (N=5 untreated; N=9 treated). Immunoblots shown above the histogram are representative images of the quantified data.  76  Figure 3.11. The protein folding chemical chaperone DMSO does not significantly rescue the expression levels of mutant containing GABA-A receptors.  DMSO  Mut -  Wt -  Wt +  Mut +  1  14000  *  12000  NS  10000 8000 6000 4000 2000  0 Wt DMSO  -  77  Mut  Wt  Mut  -  +  +  treated (8,371 ± 489) versus untreated (6,371 ± 1,034) conditions, which did not reach statistical significance (N=9 treated, N=5 untreated, unpaired t-test P=0.07).  3.2.7 The A322D mutation enhances the degradation rate of GABA-A receptors  The findings that (i) there is an increased association of the mutant receptor to calnexin and (ii) that DMSO may partially assist in correct folding of this receptor, both point to the mutation leading to increased degradation early in the biogenesis of the receptor. To investigate synthesis associated degradation more directly and to quantify any differences observed I decided to use metabolic pulse-chase experiments as these allowed me to directly assess the half-life of the wild type and mutant α1 subunits alone and in the full receptor complex. In pilot experiments I investigated the effects of different pulse times (15 – 120 min) with 35S-methionine and chase times (from 15 min up to 12 h). On the basis of these experiments, I selected 30 min as the optimum pulse time (a compromise between obtaining sufficient labeling for the experiment, but not incurring significant degradation during the pulse) and a maximum of 8 h as sufficient chase time. I also performed initial experiments to assess the optimal concentration of cycloheximide, to use to block new protein synthesis. This was necessary because continued synthesis of the receptor would artificially decrease the number of radioactive labeled α1 subunits pulled-down by immunoprecipitation since the unlabeled receptor pool would begin to outnumber the labeled receptors overtime. To quantify the effects of the mutation on the degradation rate of the α1 subunit expressed alone, I performed a 30 min pulse and examined four chase times (for 1, 2, 4, 8 h). Isotope counts for the A322D containing α1 subunit did not significantly differ from 78  wild type counts at time point zero (86 ± 15 % of control, P=0.57). This confirmed that little or no degradation differences could be observed immediately after the 30 min pulse when the mutant single subunit was expressed alone. However, I observed that the mutation had a dramatic effect on the half-life of the α1 subunit (Figure 3.12; N=3); reducing the t1/2 from 4.8 to 1.5 h (when fitted by a single exponential function between 0 to 4 h). Similarly, when cotransfected with β2γ2, there was a dramatic effect of the mutation on the stability of the α1 subunit (Figure 3.13; N=4); reducing the t1/2 from 4.4 to 1.3 h. Again, at time point zero the total counts were not statistically significantly different (70 ± 10 % of control, P=0.06). However, I did observe a trend toward greater degradation of the full receptor incorporating the A322D mutation at this early time point. Between 4 and 8 h chase time the degradation rates of the wild type and mutant subunits were similar, suggesting that the remaining fraction of the mutant α1 subunit and the assembled receptor were handled similarly to the wild type after its initial rapid degradation. At these later chase time points, doublet bands were commonly seen with mutant single subunit and, in particular, with the full receptor samples. Indeed when comparing the wild type and mutant α1 subunits visually and over multiple experiments, it appeared that the mutation may have caused an increase in the proportion of the lower molecular weight band relative to the higher molecular weight band, especially at longer chase time points (data not shown). However, this effect was technically difficult to quantify since  79  Figure 3.12. The 1(A322D) mutation reduces the half-life of the α1 subunit.  The graph plots the percent radioactivity remaining versus time for wild type (●) and mutant (▲) α1 subunits, following a 30 min pulse with 35S-methionine (N=3). The autoradiographs depicted above the histogram for wild type and mutant subunits were exposed for different lengths of time to reveal the signal at the 8 h chase point.  80  Figure 3.12. The 1(A322D) mutation reduces the half-life of the α1 subunit.  Chase (min)  0  60  120  240  480  1 1  100 80  60 40  20 0 100  200  300  Chase time (min)  81  400  500  Figure 3.13. The 1(A322D) mutation reduces the half-life of the full GABA-A receptor complex.  The graph plots the percent radioactivity remaining versus time for cells expressing the wild type (○) and mutant (∆)receptor (α1β2γ2), , respectively, following a 30 min pulse with 35S-methionine. (N=4). The autoradiograph images depicted above the histogram for wild type and mutant receptors were exposed for different lengths of time to reveal the signal at the 8 h chase point.  82  Figure 3.13. The 1(A322D) mutation reduces the half-life of the full GABA-A receptor complex.  Chase (min)  0  60  120  240  480  Wild type Mutant  100 80 60 40 20 0 100  200  300  Chase time (min)  83  400  500  the bands were too close together to be cut out separately. In addition lower molecular weight bands were often on the edge of visualization after optimizing exposure times for both wild type and mutant bands doublet bands across all time points. Based on the literature, I conjectured that the higher molecular weight band corresponded to a doubly glycosylated species and the lower molecular band a single glycosylated entity (Buller et al., 1994). To test this directly I performed experiments in which I pulsed with 0.2 µCi 35S-methionine (for 30 – 120 min) and immediately analyzed the expression of wild type and mutant α1 subunits. I found that 60 min produced an optimal labelling and therefore performed experiments using this pulse time in the presence and absence of tunicamycin, which blocks N-linked glycosylation. Individual higher molecular weight bands of wild type and mutant α1 subunits without tunicamycin treatment were estimated to be approximately 52 and 50 kDa after visual inspection of the gels at various exposure times to resolve individual bands. With tunicamycin treatment there was a loss of these higher molecular weight bands, and a concurrent increase in the appearance of a band at approximately 48 kDa (which required that the gels were overexposed for its visualization; Figure 3.14). These results confirm that higher bands correspond to single and doubly glycosylated states, respectively and they are the majority of the α1 species found early in the synthesis of the subunit. Collectively, these results demonstrate directly an increased degradation of both the α1 subunit and assembled GABA-A receptors that is caused by the A322D mutation. There is also the suggestion of an enrichment of a pool of lower molecular weight, unglycosylated, α1 receptors due to this mutation.  84  Figure 3.14. Effects of inhibiting N-glycosylation with tunicamycin.  Representative autoradiogram showing 35S-methionine labeled α1 and α1(A322D) GABA-A receptor subunits, which were anti-FLAG immunoprecipitated from transfected HEK293 cells that had been treated with (+) or without (-) tunicamycin (10 µg/ml) to block the addition of N-glycosylation carbohydrate moieties. Cells were treated (1.5 h) with tunicamycin just prior to, as well as during, the radioactive pulse (1 h). Both the 52 kDa and 50 kDa bands correspond to N-glycosylated α1 species and are the dominant forms observed under normal conditions (N=4).  85  Figure 3.14. Effects of inhibiting N-glycosylation with tunicamycin.  Tunicamycin  -  +  kDa kDa kDa  86  -  +  3.2.8 Pathways involved in the degradation of wild type and mutant α1 subunits  Since proteins that are destined for degradation are either targeted to the proteasome or the lysosome, I next examined whether the GABA-A receptors containing the A322D mutation were preferentially affected by one or the other of these degradation pathways. Firstly, to assess whether the proteasome is involved in the elimination of mutant subunits I used the proteasome inhibitor lactacystin, which specifically blocks the 20S proteasome proteolytic functions. Cells were transfected for 6 h and then incubated for 18 h with either 10 or 15 µM of the inhibitor. Under these conditions, this treatment had no effect on cell viability or morphology, as assessed visually. To confirm that the lactacystin treatment was indeed blocking the degradation of Ub-tagged proteins, I performed western blotting for poly-ubiquitinated proteins for both wild type and mutant GABA-A receptor groups (Figure 3.15). I found that both 10 and 15 µM lactacystin was sufficient to increase the proportion of ubiquitinated proteins seen, and thus, ubiquitinated GABA-A receptors would likely be accumulating in the cell under these conditions. Lactacystin significantly increased the total expression levels of both the wild type and mutant GABA-A receptors (Figure 3.16). Importantly, following proteasome inhibition, there was no longer any difference observed for mutant receptors relative to wild type untreated controls (Figure 3.16A). Analysis of percent changes of the treated versus untreated groups showed significant increases for both receptor type (Figure 3.16B). Thus, lactacystin treatment increased the total amount of wildtype subunit by 36  87  Figure 3.15. Total ubiquitination products are increased by lactacystin treatment.  Representative western blot images showing strong increases in poly-ubiquitinated proteins in lysates from wild type and mutant α1β22 cells types treated with lactacystin (10 or 15 µM).  88  Figure 3.15. Total ubiquitination products are increased by lactacystin treatment.  Mut  Wt Lactacystin (M)  0  10  100 KDa 55 KDa  25 KDa  89  15  0  10  15  Figure 3.16. The proteasome inhibitor lactacystin enhances both wild type and mutant containing GABA-A receptor expression.  A. Treatment with the 20S proteasome inhibitor lactacystin (10-15 µM) enhanced the total levels of both wild type and mutant α1β22 containing receptors expressed in HEK293 cells. The graph plots the levels of α1 expression in each condition normalized to the untreated wild type receptor (N=6). Representative western blot images are shown above the graph. B. This graph plots the percent change observed with lactacystin treatment for wild type or mutant receptors versus the untreated control levels for each receptor type (N=6). * P<0.05.  90  Figure 3.16. The proteasome inhibitor lactacystin enhances both wild type and mutant containing GABA-A receptor expression.  A Lactacystin  Mut -  Wt -  Wt +  Mut +  1  *  1.4 1.2 1.0 0.8  *  0.6 0.4 0.2 0 Wt Lactacystin  B  -  Mut  Wt  Mut  -  +  +  100  NS  *  80 60  *  40 20 0  Wild type  91  Mutant  ± 3% (N=6, P<0.05) relative to untreated wildtype receptors. Lactacystin treatment also increased the total amount of mutant subunit by 62 ± 15% (N=6, P<0.05) relative to untreated mutant receptors. These results suggest that the proteasome is an important degradation route for both the wild type and mutant receptors. An alternative route for protein degradation is via the lysosomal pathway. To investigate the possible role of this pathway I used the lysosomal protease inhibitor leupeptin. I found that when cells were incubated with leupeptin (25 µM or 50 µM) for 18 h wild type receptors were largely unaffected (Figure 3.17). In contrast this treatment resulted in an increase in the total levels of mutant receptors. However, when levels were compared to wild type untreated controls (Figure 3.17A), leupeptin treatment did not restore the expression of the mutant receptors to that of the untreated control receptors. Analysis of the percent change due to the leupeptin treatment between wild type (4 ± 6) and mutant receptor (30 ± 12%) groups showed a significant difference (Figure 3.17B; N=6, P<0.05). Thus, mutant containing receptors seem to be preferentially targeted for lysosomal degradation. Overall, the A322D mutation resulted in a GABA-A receptor that was degraded by both proteasomal and lysosomal sensitive routes. This differed from the wild type GABA-A receptors, which seemed to be degraded selectively by the proteasomal pathway.  92  Figure 3.17. The lysosomal peptidase inhibitor leupeptin preferentially blocks degradation of the α1(A322D) containing GABA-A receptors.  A. Treatment with the lysosomal peptidase inhibitor leupeptin (25-50 µM) preferentially enhanced the total levels of mutant (Mut) but not wild type (Wt) α1β22 containing receptors. The graph plots the levels of α1 expression in each condition normalized to the untreated wild type receptor (N=6). B. This graph plots the percent change observed with leupeptin treatment for wild type or mutant receptors versus the untreated control levels for each receptor type (N=6). * P<0.05, ** P<0.01.  93  Figure 3.17. The lysosomal peptidase inhibitor leupeptin preferentially blocks degradation of the α1(A322D) containing GABA-A receptors.  A Leupeptin  Wt -  Mut -  Wt +  Mut +  1  1.2 1.0  **  0.8  *  0.6 0.4 0.2 0 Wt Leupeptin  -  Mut  Wt  Mut  -  +  +  B  *  50  *  40 30 20 10 0  Wild type 94  Mutant  3.2.9 The early endosome marker Rab4 colocalizes with A322D mutant containing GABA-A receptors  While these western blotting and degradation blockade experiments demonstrate an increase in the degradation of mutant α1 subunits and mutant GABA-A receptors, the possibility remains that the mutation causes other alterations such as decreased surface receptor stability, in addition to enhanced degradation after protein synthesis. Indeed, the apparent preference for degradation of the mutant receptor by the lysosomal pathway suggests the possibility that mutant receptors are expressed on the plasma membrane but are less stable, due to enhanced endocytosis (Kittler et al., 2004). Internalizing vesicles are fused with early endosome vesicles/tubules and the proteins are resorted into vesicles bound for degradation or recycling back to the surface. To test for the possibility of enhanced endocytosis, I performed double labeling immunocytochemistry to examine the distribution of early endosomes in HEK293 cells when expressing either mutant or wild type (α1β22) GABA-A receptors (Figure 3.18). The early endosome marker Rab4 showed stronger colocalization with the mutant GABA-A receptors than with the wild type receptor. In wild type transfected cells, early endosomes appear largely in two locations: peripheral to the nucleus and close to the plasma membrane. This was seen in wild type transfected cells regardless of whether the cell morphology was rounded or more differentiated. The extent and pattern of colocalization of wild type receptors and the endosomes that occurs in HEK293 cells is consistent with previous observations of constitutive recycling for this receptor (Connolly et al., 1999). In contrast, mutant receptors showed very strong localization to endosomes,  95  Figure 3.18. Early endosomes appear more abundant, and are colocalized with mutant GABA-A receptors.  Double label immunocytochemistry, in transfected permeabilized HEK293 cells, was used to assess the co-localization of wild type (122) or mutant (1′22) GABA-A receptors (anti-1; green) with early endosomes (anti-rab4; red). The wild type merged image shows some co-localization of the wild type GABA-A receptor with the endosome marker in the intracellular region and at the membrane surface. Mutant GABAARs, however, are largely co-localized with the endosomes and both mutant receptors and endosomes are markedly more diffuse in their distribution. Scale bar = 20 m.  96  Figure 3.18. Early endosomes appear more abundant and are colocalized with mutant GABA-A receptors.  Anti-1  Anti-rab4  Merged  Wild type  Mutant  20 m  97  which were distributed much more diffusely throughout the cell. Since Rab4 is important in short range recycling between the cell surface and internal compartments, this redistribution to more intracellular locations suggests that mutant receptors are more likely to be internalized than recycled back to the cell surface.  3.2.10 Blocking dynamin-dependent endocytosis enhances the total expression of mutant GABA-A receptors  The previous immunocytochemistry experiment suggested that there may be alterations to the surface stability of the mutant GABA-A receptors such that there is an increase in the internalization of these receptors. Thus, I next explored whether interfering with specific internalization pathways may alter the total amount of these receptors as well as their surface expression levels. The major form of constitutive endocytosis for GABA-A receptors is dynamin and clathrin-dependent (Herring et al., 2003; Kittler et al., 2000). Dynamin, a guanosine triphosphatase, is a key component of the clathrin-dependent endocytic machinery, which mediates scission events of these internalizing vesicles (Mettlen et al., 2009; Mukherjee et al., 1997) Here I inhibited endocytosis using a dominant negative construct of dynamin 1 (K44E) and compared this to cells transfected with wild-type dynamin 1 (Dyn), which does not alter endocytosis (Herskovits et al., 1993; Man et al., 2000) for wild type and mutant receptor types. Within receptor types, blocking dynamin-dependent endocytosis resulted in a minor, but significant, increase in the total expression of wild type α1β2γ2  98  receptors (6,550 ± 173 arbitrary OD units for the K44E mutant versus 6,039 ± 284 arbitrary OD units for the Dyn controls). In contrast, it had a bigger effect on the mutant receptors (4,059 ± 418 OD units for the K44E mutant versus 2,904 ± 273 OD units for the Dyn controls). Analysis of the effects, relative to the wild type receptor with control wild type dynamin as the standard, showed that blocking dynamin receptor endocytosis could not rescue the mutant receptor to wild type levels (Figure 3.19A). To further examine this effect and determine an average percent change, I increased the number of experiments and assessed wild type and mutant receptors as a percent change for each experiment (Figure 3.19B). This enabled me to include experiments for which I had data for within the receptor group (wild type or mutant), but not between the two receptor groups. When examined for a percent change caused by blocking dynamindependent endocytosis, the effect on the mutant receptor (52 ± 17%; N=8, P<0.05), was much greater than on the wild type receptor (8 ± 3%, N=7, P<0.05) and statistically significant (P<0.05).  3.2.11 The cell surface expression of mutant GABA-A receptors is also increased after blockade of dynamin-dependent endocytosis  Since dynamin 1 also mediates some internal vesicle trafficking, it is possible that the total expression increases observed earlier are a result of blocking trafficking/degradation pathways prior to cell surface expression. Therefore, I also examined the cell surface expression of mutant containing receptors after dynamin blockade. I found, using cell surface biotinylation experiments, that the dominant  99  Figure 3.19. Blocking dynamin-dependent processes enhances the total expression of α1 and α1(A322D) containing receptors.  A. Co-expression of a dynamin dominant negative (K44E) was compared to coexpression of wild type dynamin (Dyn) in HEK293 cells with wild type or mutant containing GABA-A receptors using western blotting. K44E increased the expression of mutant α1-containing GABA-A receptors and to a lesser extent wild type GABA-A receptors. The graph plots the levels of α1 expression in each condition relative to wild type α1β22 co-expressed with wild type (N=5). A representative immunoblot is shown above the histogram. B. This graph plots the percent change observed for α1 subunits with the K44E versus Dyn controls for wild type receptor (N=7) and mutant receptor (N=8) groups. * P<0.05, *** P<0.001.  100  Figure 3.19. Blocking dynamin-dependent processes enhances the total expression of α1 and α1(A322D) containing receptors.  A  Wild type  1  Dyn  Mutant Dyn  K44E  K44E  1.4 1.2  *  1.0  *  0.8 0.6  ***  0.4 0.2 0 Dyn  Wild type  B  K44E  Dyn  K44E  Mutant  *  80  * 60  40  20  * 0  Wild type  101  Mutant  negative form of dynamin increases the cell surface levels of α1(A322D) containing GABA-A receptors (5083 ± 806 OD units for the K44E mutant versus 4372 ± 814 OD units for Dyn controls). While the effect was relatively small overall in these experiments, this was probably due to higher variability, and was statistically significant with a paired Student t-test. Nonetheless, mutant values were still significantly less relative to wild type receptor controls (Figure 3.20A; N=4). Under these conditions, I did not find a change in the wild type receptor’s cell surface expression with the K44E mutation (10,200 ± 748 OD units) relative to wild type control (10,465 ± 807 OD units). Again, to further examine this effect, I assessed wild type and mutant receptors as a percent change for each group (Figure 3.20B). Here I included additional experiments for which I only had data for one or the other group. When examined for a percentage change caused by blocking dynamin-dependent endocytosis, the effect on the mutant receptor (23 ±8 %; N=6, P<0.01), was much greater than on the wild type receptor (1 ± 1%; N=5). The difference between the effect of the dominant negative on wild type and mutant receptors was statistically significant (P<0.05). These findings further confirm that mutant receptors have enhanced cell surface endocytosis, which is mediated by a dynamin-dependent pathway.  3.2.12 The total expression of mutant GABA-A receptors is increased after blockade of caveolin 1  The effects of the dynamin dominant negative were relatively modest. This might have been because of incomplete block of dynamin-dependent endocytosis. However,  102  Figure 3.20. Blocking dynamin-dependent endocytosis preferentially enhances the surface expression of α1(A322D) containing receptors.  A. Co-expression of a dynamin dominant negative (K44E) was compared to coexpression of wild type dynamin (Dyn) in HEK293 cells for wild type and mutant containing GABA-A receptors after surface biotinylation and western blotting. K44E increased the expression of mutant α1 containing receptors, but did not affect wild type surface receptor numbers. The graph plots the levels of α1 expression in each condition relative to wild type α1β22 co-expressed with wild type dynamin (N=4). A representative immunoblot is shown above the histogram. B. This graph plots the percent change of surface expression of GABA-A receptors resulting from expression of the K44E mutant (relative to the Dyn control) for wild type receptor (N=5) and mutant receptor (N=6) groups. * P<0.05, *** P<0.001.  103  Figure 3.20. Blocking dynamin-dependent endocytosis preferentially enhances the surface expression of α1(A322D) containing receptors.  A  Wild type Dyn  1  K44E  Mutant Dyn  K44E  1.4 1.2 1.0 0.8 0.6  *** ***  0.4 0.2 0 Dyn  Wild type  B  K44E  Dyn  K44E  Mutant  50  *  40  *  30 20 10 0  Wild type 104  Mutant  when I increased the amount of the dynamin mutant expressed this resulted in toxicity and cell death. Another explanation for the incomplete block of endocytosis is that there are clathrin-independent processes involved (Cinar and Barnes, 2001). I therefore wondered whether the stability of A322D mutant-containing GABA-A receptors may also be affected by the raft restricted, caveolin 1-dependent mechanism (Nabi and Le, 2003). I, therefore, performed experiments using a dominant negative of caveolin 1. This construct consisted of caveolin 1 containing GFP tagged to the N-terminus (CavDN), which has previously been demonstrated to block endocytosis of SV-40 virus via caveolae formation in caveolin 1 containing cells (Helenius and Aebi, 2004). I examined both the total as well as surface expression levels of wild type and mutant containing GABA-A receptors. Inhibition of caveolin 1-dependent endocytosis tended to increase the total expression of wild type receptors (12,995 ± 1,087 OD units) compared to GFP controls (11,242 ± 1,384 OD units) though this effect did not reach significance (N=4, P=0.14). In contrast, the Cav-DN had a significant effect on the total expression levels for mutant receptors (6,714 ± 1,098 OD units) compared with GFP controls (4,363 ± 930 OD units; N=6, P<0.01). However, with respect to wild type receptors expressed with GFP controls, mutant receptors expressed in Cav-DN expressing cells were still reduced (Figure 3.21A). When I analyzed the percent increase in mutant receptor expression levels due to caveolin 1 inhibition there was a variable but significant effect (82 ± 29%; N=6, P<0.01; Figure 3. 21B). In contrast, the effect on the wild type receptor of this blockade was more moderate and not significant though an upward trend was observed (20 ± 10%,  105  Figure 3.21. Blocking caveolin 1-dependent endocytosis enhances the total expression of α1(A322D) containing receptors.  A. The effects on GABA-A receptor total expression with the co-expression of a caveolin 1 dominant negative (Cav-DN) was compared to co-expression of GFP in HEK293 cells for wild type and mutant-containing GABA-A receptors, using western blotting. The graph plots the levels of α1 expression in each condition relative to wild type α1β22 co-expressed with GFP controls (N=6). A representative immunoblot is shown above the histogram. B. This graph plots the percent change observed for CavDN expressed relative to GFP for each receptor type (N=6). * P<0.05, ** P<0.01; *** P<0.001.  106  Figure 3.21. Blocking caveolin 1-dependent endocytosis enhances the total expression of α1(A322D) containing receptors.  A  Wild type  Mutant  Cav DN  GFP  Cav DN  GFP  1 1.4 1.2 1.0 0.8  **  0.6  ***  0.4 0.2 0 Cav DN  GFP  Wild type  B  Cav DN  GFP  Mutant  * 120  **  100 80 60 40 20 0  Wild type 107  Mutant  N=6, P=0.18). The difference between the effect of the Cav-DN on wild type and mutant receptors was statistically significant (P<0.05).  3.2.13 The cell surface expression of mutant GABA-A receptors is also increased after blockade of caveolin-dependent endocytosis  Since it was unclear whether caveolin 1 may, like dynamin, have other functions in the cell that might affect the degradation of A322D receptor intracellularly, I also sought to determine the proportion of receptors found on the cell surface. I found a similar trend in the cell surface numbers as that determined for total expression levels, following inhibition of caveolin 1-dependent function. Wild type receptors were elevated (13,583 ± 1,158 OD units) with Cav-DN expression relative to receptors cotransfected with GFP (11,599 ± 1,559 OD units) but did not reach statistical significance (P=0.36, N=4). In contrast, mutant containing receptors were significantly increased with Cav-DN expression (8,673 ± 1,069 OD units) relative to their GFP controls (5,401 ± 594; N=4; P<0.05). Interestingly, when expressed as a proportion of wild type controls, blocking caveolin-dependent endocytosis enhanced the number of surface mutant receptors such that they were no longer significantly different (Figure 3.22A; N=4, P=0.23). The percent change in α1 surface expression resulting from expression of the CavDN is plotted in Figure 3.22B and again includes additional data, which are from only one or the other group. The mutant receptor showed a significant increase of 62 ± 12% (Figure 3.22B; N=4, P<0.05). However, the wild type receptor was unchanged (10 ± 17%, N=6) under these conditions.  108  Figure 3.22. Blocking caveolin 1-dependent endocytosis preferentially enhances the surface expression of α1(A322D) containing receptors.  A. The effects on GABA-A receptor surface expression of the co-expression of a caveolin 1 dominant negative (Cav-DN) was compared to co-expression of GFP in HEK293 cells for wild type and mutant-containing GABA-A receptors, using surface biotinylation and western blotting. The graph plots the levels of α1 expression in each condition relative to wild type α1β22 co-expressed with GFP control (N=4). A representative immunoblot is shown above the histogram. B. This graph plots the percent change in GABA-A receptor surface expression resulting from Cav-DN treatment (presented relative to GFP) for wild type receptor type (N=6) and mutant receptor (N=4) groups. * P<0.05.  109  Figure 3.22. Blocking caveolin 1-dependent endocytosis preferentially enhances the surface expression of α1(A322D) containing receptors.  A  Wild type Cav DN  GFP  Mutant GFP  Cav DN  1 1.6 1.4 1.2 1.0 0.8 0.6  *  0.4 0.2 0 Cav DN  GFP  Wild type  B  Cav DN  GFP  Mutant  120 100 80  *  60 40 20 0  Wild type 110  Mutant  These results suggest that the mutant receptor is less stable on the membrane surface and is endocytosed via a caveolin 1-dependent process. Together, my study provides the first evidence for the involvement of caveolin-dependent mechanisms in GABA-A receptor trafficking, and suggests that alteration of this mechanism may, at least in part, contribute to the reduced expression of the GABA-A receptor α1(A322D) mutant.  3.2.14 A novel endocytosis pathway for the internalization of GABA-A receptors.  The results using the caveolin-1 dominant negative suggested that GABA-A receptors are internalized, at least in part, via a lipid raft-dependent, caveolin 1-dependent mechanism. I wondered, therefore, whether there might be a direct binding of GABA-A receptors to caveolin 1. Indeed, analysis of the amino acid sequence for α1-6 subunits revealed a putative classic caveolin-1 scaffold domain (CSD) in the extracellular Cterminus or putative TM4 region that is present in all α subunit types (Figure 3.23). Consistent with this hypothesis, I found that following co-transfection of HEK293 cells with the single α1 subunit and with caveolin 1 that these two proteins could be coimmunoprecipitated (Figure 3.24). Since the α1 subunit bound caveolin 1, this suggests that the association of caveolin 1 and the GABA-A receptor α subunits occurs early in the synthesis pathway and is independent of subunit oligomerization. Indeed it may also be an important targeting element for that portion of GABA-A receptors that are bound for lipid rafts as caveolins strongly bind to cholesterol and act as raft scaffolding elements as well (Hailstones et al., 1998; Lajoie and Nabi, 2007; Murata et al., 1995). 111  Figure 3.23. A putative consensus caveolin binding domain in the rat GABA-A receptor α subunits.  Clustal-W alignment of rat α (1–6) amino acid sequences showing a putative classic caveolin scaffolding domain (CSD) identified on the border of the extracellular Cterminus and transmembrane four of all the α subunits. The consensus sequences for both the classic CSD and a non-classical CSD site are show below where  (red) = any aromatic amino acid and X = any amino acid.  112  Figure 3.23. A putative consensus caveolin binding domain in the rat GABA-A receptor α subunits.  Transmembrane 4 GBRA1_rat GBRA2_rat GBRA3_rat GBRA4_rat GBRA5_rat GBRA6_rat  KIDRLSRIAFPLLFGI KIDRMSRIVFPVLFGT KVDKISRIIFPVLFAI KIDKYARILFPVTFGA KIDKMSRIVFPILFGT KIDQYSRILFPVAFAG  FNLVYWATY FNLVYWATY FNLVYWATY FNMVYWVVY FNLVYWATY FNLVYWIVY  LNREPQLKAPTPHQ LNREPVLGVSP--VNRESAIKGMIRKQ LSKDTMEKSESLMLNREPVIKGATSPK LSKDTMEVSSTVE-  Caveolin scaffolding domain classic binding motif  consensus sequences  xxxx  xx  and  x  xxxx   = any aromatic amino acid x = any amino acid  113  455 451 493 552 464 453  Figure 3.24. Co-IP of caveolin 1 and GABA-A receptor α1 subunits expressed in HEK293 cells.  Lysates of HEK293 cells transfected with the GABA-A receptor α1 subunit and caveolin 1 (Cav-1) and subjected to immunoprecipitation (IP) with a rabbit polyclonal anticaveolin 1 showed co-immunoprecipitation of the α1 subunit. The reciprocal IP with anti-FLAG for α1 subunits showed co-immunoprecipitation for Cav-1.  114  Figure 3.24. Co-IP of caveolin 1 and GABA-A receptor α1 subunits expressed in HEK293 cells.  1 Cav-1 lysate  IgG  IP: Cav-1  IB: 1 IB: Cav-1  115  GFP IP: Flag  No lysate  IP: Flag IP: Flag  CHAPTER FOUR DISCUSSION  4.1 GABA-A receptor mutations that are associated with an epileptic phenotype  In the present study I have investigated the α1(A322D) mutation of the GABA-A receptor subunit, which was one of the first mutations associated with an epileptic phenotype in humans. Since the inception of this study, a number of additional mutations have been characterized in various subunits of the GABA-A receptor including the 2, , β3 and the α1 subunits (Figure 4.1). These mutations are associated with a variety of epileptic phenotypes such as GEFS+, childhood absence epilepsy as well as juvenile mycolonic epilepsy (For a recent review see Macdonald and Kang, 2009). The highest frequency of mutations occurs in the 2 subunit where at least 7 different point mutations have been isolated while other subunits, especially the α1 subunit, have yielded relatively few tolerated mutations associated with the disease. Mutations of the GABA-A receptor subunit genes generally fall into one of three groups: 1) missense, 2) nonsense, and 3) frameshift mutations. The α1(A322D) mutation studied here results from a missense mutation, which alters the nucleotide sequence of the codon resulting in a change of amino acid in the final peptide from an alanine to an aspartate. In general, the effects of a missense mutation can be highly variable depending on how different the new amino acid is from the original. Some missense mutations have no effect perhaps because the replacement is a similar amino acid or the region is relatively tolerant of the change,  116  Figure 4.1 GABA-A receptor mutations associated with epilepsy. (Modified from Macdonald and Kang, 2009.)  aa α  11 α1(A322D) 12 α1(S326fs328X)  β  1 β3(P11S) 2 β3(S15F) 4 β3(G32R)  γ  δ  3 5 6 9 10 13 14  g2(Q1X) g2(R43Q) g2(R139G) g2(IVS6+2T>G) g2(K289M) g2(Q351X) g2(W390X)  1 2 3  5  4  6  8 9  10  TM1  11 12  7 δ(E117A) 8 δ(R220H/C) 13  117  TM4 14  7  while others can increase susceptibility to a disease or are strongly associated with a disorder. Many of the GABA-A receptor subunit mutations impair surface expression of the receptor and/or have reduced channel function. One of the earliest GABA-A receptor mutations linked to childhood absence epilepsy and febrile seizures was found in a large Australian family with a mutation in the diazepam binding site of the 2 subunit (R43Q) (Baulac et al., 2001). Expression of the 2(R43Q) mutant, in HEK293-T cells resulted in reduced peak current (Bianchi et al., 2002) and surface receptors (Kang and Macdonald, 2004).  In 2(R43Q) knock-in mice cortical pyramidal neurons from layer 2/3 showed  significantly reduced current amplitude (Tan et al., 2007). Consistent with previous results in heterologous cell lines (Sancar and Czajkowski, 2004), the mutation also caused a reduction of surface GABA-A receptors containing the 2(R43Q) subunit (Tan et al., 2007). The mutation alters diazepam modulation and GABA binding possibly by perturbing intersubunit contacts (Goldschen-Ohm et al., 2009; Hales et al., 2005) or assembly of receptors (Frugier et al., 2007). The use of transcranial magnetic stimulation has allowed the assessment of cortical inhibition in human brains due to this R43Q mutation (Fedi et al., 2008). These data confirm that cortical excitability is increased in affected subjects relative to their unaffected family members. Other mutations that show reduced cell surface expression of receptors include the  E177A and R220H mutants, which confer a susceptibility to a complex epileptic phenotype (Feng et al., 2006) and show altered channel gating. A nonsense mutation which produces a C-terminally truncated receptor 2(Q351) has also been shown to have  118  ER retention, reduced cell surface expression (Harkin et al., 2002) and acts as a dominant negative on the wild type receptor when co-expressed (Kang et al., 2009). The α1(A322D) subunit mutation is a missense mutation found within TM3 that replaces the uncharged small amino acid alanine with the large highly negatively charged aspartate. The first analysis of this mutant receptor, expressed in HEK293 cells, found a reduced sensitivity to GABA in whole cell recordings, but no change in protein expression (Cossette et al., 2002). This suggested that the primary deficit was either a reduction in GABA surface expression or a reduction in the function of receptor expressed at the surface. However, given that transmembrane domains are typically rich in hydrophobic amino acids, I expected that the introduction of a charged amino acid in this domain would have profound effects on the ability of the subunit to achieve its appropriate conformation in the ER. I therefore re-examined the effects on total GABA expression as well as investigated possible mechanisms by which the mutation could impair the trafficking of the receptor.  4.2 Summary of the results In this work I have shown that the α1(A322D) containing GABA-A receptor has reduced cell surface expression. I have provided evidence that the A322D point mutation leads to the production of a GABA-A receptor that is less stable than its native counterpart due to enhanced degradation. Mutant subunits and receptors were found to degrade relatively quickly after synthesis through ER associated degradation. The mutant receptor was not significantly affected by lowering the incubation temperature to enhance 119  proper folding conformations, but there was a trend towards a moderate enhancement with the chemical chaperone DMSO. Interestingly, mutant subunits were capable of oligomerization with β2 and 2 subunits. These subunits formed a trafficking competent receptor which was less stable on the cell surface due to enhanced endocytosis. Degradation of the mutant receptor occurs preferentially through lysosomes and endocytosis of the mutant receptor was both dynamin and caveolin dependent. I further present evidence that the GABA-A receptor α1 subunit, which contains a classic caveolin-1 binding domain in all members of the subunit class, co-IPs with caveolin-1 and vice versa suggesting this may be a common mechanism for GABA-A receptor endocytosis.  4.3 Changes in the surface expression of the α1(A322D) mutant Mutant GABA-A receptors expressed in HEK293 cells showed significantly less cell surface distribution and much greater intracellular signal relative to wild type expressing cells. Since homomeric α1 receptors do not reach the cell surface (Connolly et al., 1996) it can be concluded that receptors containing the mutant subunit can be assembled with β and  subunits and are able to be targeted to the plasma membrane. The reduction in the number that were surface expressed could be because fewer subunits are assembled and targeted to the cell surface and/or because the receptor is less stable on the membrane surface.  120  4.4 Changes in total expression of the α1(A322D) mutant There is disagreement whether the mutation affects the total amount of α1 subunits and hence the amount of receptor available for insertion into the membrane (Cossette et al., 2002; Gallagher et al., 2007; Gallagher et al., 2004). In an earlier study from our laboratory, the preliminary analysis of the total expression levels suggested there was no change. However, in a more quantitative analysis we reported a significant reduction of the mutant receptor (Bradley et al, 2004 Soc Neurosci Abstr 567.11) as have other studies (Gallagher et al., 2007; Gallagher et al., 2005; Gallagher et al., 2004). The results presented in this thesis comprise the data presented in Bradley et al (2004) and studies performed subsequently, all of which showed a decrease in total expression. Therefore it can be concluded that the mutation results in a substantial reduction in the total amount of receptor. The decrease in total expression could be due to a decrease in protein synthesis, an increase in degradation or both.  4.5 The mutant receptor shows enhanced misfolding Misfolding rates for wild type GABA-A receptor subunits are very high (Buller et al., 1994; Gorrie et al., 1997) and so mechanisms exist to degrade errant subunits via ERAD. Given that the 1(A322D) mutation introduces a negative charge into the middle of the third transmembrane domain of the 1 subunit it would seem plausible that the mutation results in increased misfolding rates of the subunit. In support of this idea, I  121  found that there was an increased association of the ER chaperone calnexin with the mutant compared to the wild type subunits. Calnexin binds to GABA-A receptor subunits within the ER (Connolly et al., 1996), where it retains misfolded subunits (Helenius and Aebi, 2004; Moremen and Molinari, 2006; Parodi, 2000). Thus, the increase in association with calnexin suggests that a high proportion of mutant subunits are retained within the ER and subsequently targeted for degradation. Evidence from other studies on the α1(A322D) subunit have also implicated misfolding and ERAD as the mechanistic reason for the epileptic phenotype in humans (Fisher, 2004; Gallagher et al., 2007; Gallagher et al., 2004). Gallagher and colleagues showed similar reductions of total expression of both the homomeric α1(A322D) receptor and α1(A322D) expressed with β22 subunits in HEK293 cells with western blotting (Gallagher et al., 2004). Gallagher et al, propose that degradation occurs prior to assembly with the other subunits and that approximately 33% of the α1(A332D) subunit is misfolded such that TM3 is unable to incorporate into the lipid bilayer. The way in which the authors determined this was by mutating all the leading glycosylation sites and looking for formation of a second band if a constructed N-glycosylation site (between TM3 and TM4) was shifted from the cytoplasmic side to the ER luminal side. They then observed one molecular weight species for the wild type α1 subunit and measured the appearance of a second band in the α1(A322D) subunit. They concluded that the α1(A322D) mutation caused failure of lipid insertion. Because the proportion of subunit that failed to achieve appropriate topology didn’t account for the dramatic loss of the total α1 expression, the authors also propose that other misfolding problems may occur possibly due to inappropriate stacking of the amino acids as they coil and form the  122  membrane helix. Consistent with these results, I have provided evidence that theA322D mutation causes ER retention of a misfolded α1 subunit through enhanced calnexin binding independent of coexpression with other subunits. Since, multiple cycles of deglycosolation /reglycosylation and prolonged calnexin binding have been shown to be important intermediate triggers for a protein to be identified as an ERAD target (Helenius and Aebi, 2004; Kleizen and Braakman, 2004; Raasi and Wolf, 2007; Richly et al., 2005), it can also be concluded that a greater proportion of these subunits will likely also have enhanced degradation through the proteasome.  4.6 Effects of temperature and DMSO on the expression of mutant GABA-A receptors  Temperature may alter the rate at which a protein can obtain its most stable configuration. High temperatures can cause energetically costly interactions to occur more frequently and cause misfolding. Lowering the temperature in contrast, slows the reaction down such that energetically favorable reactions, often those that have high stability, may be obtained. A number of mutations in various proteins such as cystic fibrosis transmembrane conductance regulator (CFTR; Denning et al., 1992) have been determined to be temperature sensitive (Brown et al., 1997). However, a number of the mutant α1(A322D) receptors were still reaching the cell surface, so I sought to determine if lowering the incubation temperature might permit the accumulation of mutant receptors that would escape ERAD. I examined the total expression of the mutant receptor at 30C relative to the wild type receptors. I found that the proportion of expressed mutant  123  receptor was unchanged relative to wild type receptor expression. Under these conditions, the misfolding of the receptor appeared insensitive to a reduction in temperature. Similarly, high temperature incubation (40 C for one hour) does not further destabilize peak current amplitude in heterozygous α1(A322D)β22S receptors relative to currents observed at 37C (Kang et al., 2006a). To further characterize the mutant receptors, I treated mutant α1(A322D) containing receptors with optimized levels of DMSO, that has previously been shown to act as a chemical chaperone. It has been suggested that DMSO may act as an osmolyte and interact with proteins, but the specific mechanism for enhancing folding rates is unclear (Papp and Csermely, 2006). Again, the α1(A322D) mutant receptor, could not be fully rescued though a trend towards greater stability was observed.  4.7 Protein degradation rates are increased by the 1(A322D) mutation. I found an enhanced degradation of mutant α1 subunits using pulse-chase experiments. Broadly similar results were published by another group during the course of this thesis (Gallagher et al., 2007). In my experiments, there were some quantitative differences from this previous report. In particular, I observed a much longer half-life for the mutant α1 subunit of approximately 100 min, compared with an estimated 23 min in this other study. One possible explanation for this difference was that I included a protein synthesis inhibitor in the chase medium to prevent synthesis of new unlabeled subunits, which might otherwise interfere with the efficiency of the co-IP of radiolabelled α1 subunits. Also, by extending the chase time of my experiments to 8 hours (as opposed  124  to 180 min in the previous study) I was also able to directly measure the half-life of the native α1 subunit, which was approximately 260 min and was consistent with earlier analyses of neuronal GABA-A receptor half-life of approximately 3.8 h (Borden and Farb, 1988; Czajkowski and Farb, 1989). Therefore, in my hands the mutation increased the degradation rate of the α1 subunit by approximately 2.5 fold. I also compared the degradation rates of the α1β2γ2 combination, to see whether the assembled receptor was degraded differently when it contained a mutant subunit. In wild types, there was a difference in the degradation profiles of α1 subunits between cells expressing the subunit alone or in combination with β2 and γ2. This was most noticeable after 4 h, where the α1 subunit was degraded by about 50% when expressed alone and by approximately 35% when expressed in combination with the other subunits. This suggests that a significant fraction of the α1 subunits are co-assembled with β2 and γ2 subunits and, as a result, their degradation rate is slowed in the early phase of biogenesis. However, when I compared the degradation rates of the wild type and mutant receptor I observed a similar difference in decay time constant as when I studied the isolated subunit. Thus, the co-assembly of the subunits into a trafficking competent receptor, which occurs rapidly within the ER (Connolly et al., 1996), is not a dominant factor in determining the degradation rate of the mutant α1 subunit prior to export to the cell surface. Synthesis of the α1 subunit also appeared similar to wild type levels at T=0 under these conditions suggesting that the rate of synthesis of the α1(A322D) subunit was unaffected by the mutation. This agrees with a previous report where no change of  125  synthesis rates was found between wild type and mutant subunit expression at short pulse times (Gallagher et al., 2007).  4.8 The mutation is associated with a reduction in glycosylated subunits The extent to which a receptor is glycosylated may alter both its localization and its function. Recently, hyperglycosylation has been linked to remitting childhood absence epilepsy in a large scale study of GABRB3 mutations (Tanaka et al., 2008) . Though the mechanism is unclear, deficits in receptor function are evident in a drastic drop of evoked current density in HEK293 cells expressing the P11S, S15F and G32R mutations. Loss of cell surface receptors has also been shown for α1 glycosylation sites Q10 and Q110 which were point mutated to alanine (Buller et al., 1994). Interestingly, [3H]Ro15-1788 binding returned to wild type levels when transfected HEK293 cells (α1,β12 ) were grown at 30C suggesting a temperature sensitivity of the mutations and a restoration of correct folding. In addition to enhanced degradation, the mutation also appeared to reduce the proportion of glycosylated subunits in both the α1 subunits and the α1β2γ2 assemblies most likely through the enhanced calnexin binding. Since the mutation does not affect the N-terminal domain where the carbohydrate moieties would have attached, the reduction of glycosylated 1(A322D) subunits is most likely due to the increased targeting of these subunits to the ER degradation pathway and suggests that unglycosylated, immature receptors may be the mutant receptor type that reaches the cell surface.  126  4.9 The role of ERAD in the degradation of mutant α1(A322D) GABA-A receptors  Surface expression levels of GABA-A receptors can be regulated by the proteasome at the ER level. Under constitutive conditions, poly-ubiquitinated GABA-A receptors in the ER have been shown to be stabilized by association with Plic-1 and bind to α1 subunits (Bedford et al., 2001; Saliba et al., 2008). Plic-1’s role may be to enhance the rate of secretion by blocking the proteasomal degradation of ER retained receptors, thus increasing the number of receptors on the cell surface (Saliba et al., 2008). Similarly, the related neuronal nicotinic acetylcholine surface receptor number (Rezvani et al., 2009) is also modulated by the ubiquitin-proteasome. The authors found that treatment of PC-12 cells expressing nAChRs with the proteasome inhibitior PS-341 caused the accumulation of the receptor in the ER and golgi fractions, suggesting that ERAD through the ubiquitin-proteasome pathway was the primary route for altering surface receptor numbers. Furthermore, activity-dependent changes for GABA-A receptor surface expression is also regulated by ER related degradation that is dependent on proteasomal function. Saliba and co-workers showed in cortical neurons that in the presence of TTX to block activity, β3 subunits had increased amounts of Ub conjugates and reduced cell surface expression. This loss of GABA-A receptors occurred in the presence of Brefeldin A, which blocks ER to golgi transport, strongly implicating ERAD mechanisms in the activity dependent down regulation of GABA-A receptor function. Furthermore, co-treatment of neurons with TTX and epoxomicin to block proteasome  127  function showed that epoxomicin occluded the effect of TTX on cell surface receptor numbers indicating that downregulation was dependent on proteasomal degradation of the receptor. Here I found that the ubiquitin proteosome complex, is involved in the degradation of both native and mutant receptors. The time frame of the pulse-chase data and the early loss of significant numbers of both mutant and wild type subunits and receptors strongly suggest that ERAD is important for both the wild type and mutant form of the receptor’s early degradation. The vast majority of ER detained proteins are subject to polyubiquitination, retrotranslocation to the cytosol and degradation by the proteasome machinery. In this study lactacystin inhibition of the proteasome blocked degradation of both the wild type and the mutant receptor. Interestingly, from my pulse-chase data at early time points, when it is expected that the receptor has yet to be transported to the cell surface (less than 6 h expression) (Gorrie et al., 1997), I observed that the wild type receptor is also subject to significant levels of misfolding and/or misassembly which could then led to its subsequent removal from the receptor pool by ERAD. This correlated well with the percent change in total amount of expressed receptor detected after treatment with lactacystin. My results are in partial agreement with an independent study of the α1(A322D) subunit expression alone (Gallagher et al., 2007). Here lactacystin treatment did not lead to an increase in mutant receptor levels sufficient to equal those of wild type receptors indicating there was a partial recovery, but not a full recovery. In the report from Gallagher et al, mutant subunits also did not recover to wild type levels in the presence of the proteasomal inhibitor lactacystin so it was surprising that they did not consider other  128  routes of degradation. However, they found that mutant subunits were more polyubiquitinated than wild type α1 subunits based on the ratio of ubiquitination of the immunoprecipitated subunits with or without lactacystin treatment. Based on this observation they concluded that the mutant homomeric receptor was preferentially degraded by the proteasome. In my experiment, however, there was no significant difference between the extent of degradation for mutant compared to wild type receptors by this pathway. The primary difference between my results and these of Gallagher et al, is that I have included the β2 and 2 subunits and so the α1 mutant subunit is capable of assembling and forming a trafficking receptor and is then subject to other degradation mechanisms. Thus, for the trafficking competent receptor, the extent to which mutant subunits are preferentially targeted to the ERAD pathway is unclear.  4.10 The role of lysosomal degradation of mutant α1(A322D) GABA-A receptors  Upon endocytosis from the plasma membrane and transfer through the early and late endocytic vesicles, many receptors and ligands may either be recycled back to the cell surface or transferred to lysosomal vesicles for proteolyic cleavage and break down into constituent amino acids as a part of the down-regulation of signaling (for a recent general review of degradation see Knecht et al., 2009; Miaczynska et al., 2004; Mukherjee et al., 1997). Lysosomes are vesicles made up of acid dependent hydrolases that work effectively at pH 4.8. Hydrolyases in the lysosome can breakdown proteins,  129  nucleic acids, lipids, organelles, bacteria and viruses to protect the cell as well as maintain the protein and lipid homeostasis of the cell. Multiple methods exist to direct receptors and their ligands into recycling or degradative pathways. For example, monoubiquitination at lysine residues on the target protein or poly-ubiquitination chains with Ub linkages at residues such as Lys63 of the Ub protein mediates the internalization of membrane receptors and targets them to lysosomes (Galan and Haguenauer-Tsapis, 1997; Roth and Davis, 2000; Shih et al., 2000). In addition, a receptor may be tagged with polyubiquitin that directs it for internalization and breakdown by the proteasome. This is true of many post synaptic density proteins (Ehlers, 2003) such as AMPA receptors, which have been shown to be regulated by both the ubiquitin-proteasome degradation pathway at the postsynaptic densities (Patrick et al., 2003) and lysosomes (Lee et al., 2004). In contrast, other receptors such as the acetylcholine receptor, seem to be largely directed to late endosomes for lysosomal degradation after endocytosis (Clementi et al., 1983; Darsow et al., 2005; Hyman and Froehner, 1983; Kumari et al., 2008). It has been suggested that GABA-A receptors that are endocytosed from the plasma membrane are primarily degraded by a leupeptin-sensitive route (Kittler et al., 2004). In contrast to my experiments with the proteasome inhibitor lactacystin, treatment with leupeptin did reveal a much larger degradation of mutant compared with wild type receptors. This suggests that mutant receptors are preferentially degraded via the lysosomal pathway. Therefore, my data was consistent with an increase in degradation of mutant receptors which have been trafficked to and then removed from the plasma membrane.  130  4.11 The role of dynamin-dependent endocytosis in the trafficking of wild type and mutant GABA-A receptors  Having found a preferential effect for lysosomal inhibition on the mutant α1(A322D) containing receptor, I looked at the role of endocytosis more directly. I first examined the distribution of mutant and wild type receptors relative to the proportion of Rab4 positive immunostaining. I found that intracellular mutant receptors were strongly colocalized with early/sorting endosomes. However, the distribution was such that the intracellular localization could only suggest that endocytosis may be upregulated here so I turned to methods that allowed me to directly examine endocytosis. Many membrane bound proteins, including several ligand-gated receptors (Carroll et al., 1999), are internalized by clathrin-mediated endocytosis which require AP2/Eps 15 interacting proteins (Benmerah and Lamaze, 2007; Benmerah et al., 1998). Since endocytosed GABA-A receptors could be observed in clathrin-coated pits (Tehrani and Barnes, 1993), clathrin-mediated endocytosis has been proposed to be a primary candidate mechanism responsible for the internalization of cell-surface GABA-A receptors through its direct binding to AP2 (Herring et al., 2003; Kittler et al., 2000). Functionally, clathrin-mediated endocytosis is important for constitutive endocytosis of GABA-A receptors as a rapid change in cell-surface numbers could be achieved by interfering with clathrin-dependent endocytosis using a peptide of β3 carrying the atypical AP2-binding motif or dynamin-amphiphysin interfering peptides (Kittler et al., 2000). By the use of dominant negative constructs of dynamin, it has been shown that this GTPase, which is involved in the budding off of internalizing clathrin coats  131  (Ungewickell and Hinrichsen, 2007), is likewise important in GABA–A receptor internalization (Herring et al, 2003; Lajoie and Nabi, 2007). Here I have found that interfering with dynamin, using expression of the K44E mutant, had only a small effect on the total and surface expression levels of wild type α1 subunits. This may reflect the relative stability of GABA receptors expressed on the surface of HEK293 cells or a partial blockade. In contrast, the dynamin mutant had a greater, though more variable, effect on the total and surface levels of mutant α1 subunits, suggesting that receptors containing this mutant subunit are more susceptible to dynamindependent endocytosis. On the basis of these observations I suggest that mutant containing receptors that escape ERAD are being inserted into the plasma membrane, but then are readily endocytosed and broken down. In contrast, wild type receptors are expressed and stabilised on the plasma membrane and this fraction contributes significantly to the total levels of receptors measured in the western analysis. Consistent with this hypothesis, I have found that the dynamin dominant negative increases the amount of mutant receptor expressed on the plasma membrane, using surface biotinylation.  4.12 The role of caveolin-dependent endocytosis in the trafficking of wild type and mutant GABA-A receptors  Several lines of evidence have shown that there are multiple endoyctic routes for the internalization of various proteins, in addition to clathrin-dependent endocytosis (for recent reviews see Hansen and Nichols, 2009; Lajoie and Nabi, 2007). These poorly  132  defined routes are generally classed as non-clathrin dependent endocytosis for lack of a better knowledge about the important mediators involved (Hansen and Nichols, 2009). Caveolae are smooth, non-clathrin coated PM invaginations, of 50-80 nm in diameter, at the membrane surface (Nichols, 2002) and are cholesterol and sphingolipid enriched (Stan et al., 1997). Caveolae have been found to be enriched with caveolin family proteins (caveolin 1-3). Caveolin-1 is expressed in non-striated muscle cells while caveolin-3 is expressed in skeletal and cardiac muscle cells and drive the formation of caveolae in each tissue type, respectively (Drab et al., 2001; Rothberg et al., 1992; Way and Parton, 1995). Due to their sensitivity to cholesterol depletion, caveolae are considered to be constituents of the lipid raft microdomains of the plasma membrane (Nabi and Le, 2003). Caveolae are suggested to form at the Golgi complex, which involves the oligomerization of caveolin and cholesterol binding, and are exported and fuse to the PM (Tagawa et al., 2005). At the PM, caveolae are relatively stable structures, but can be endocytosed to be recycled or fuse with the cavesome (caveolin-1 positive endocytic vesicles) in a kiss and run fashion (Pelkmans and Zerial, 2005) that does not involve a breakdown of the caveolae (Kirkham et al., 2005; Pelkmans et al., 2001; Tagawa et al., 2005). Interestingly, this process is dependent on dynamin 2. Dynamin is involved not only in clathrin-dependent endocytosis but also in clathrin-independent, cholesterol-dependent endocytosis from lipid rafts (Henley et al., 1998; Lajoie and Nabi, 2007; Oh et al., 1998) and is strongly colocalized with caveolae (Henley et al., 1998). Dynamin-dependent raft pathways involve the endocytosis of caveolae, which are enriched in caveolin-1. This protein is essential for the formation of  133  caveolae, and so interference with its function can be used to prevent caveolae/raft dependent endocytosis (Figure 4.2). Since GABA-A receptors have been reported to be resident in the lipid raft fractions of neurons (Dalskov et al., 2005) I also investigated the role of this form of endocytosis. My findings resembled my earlier observations with the dynamin dominant negative on total expression, suggesting that endocytosis of GABA-A receptors from HEK293 cells is predominantly mediated by a dynamin-dependent, caveolin-dependent mechanism. Interestingly, analysis of the surface expression with caveolin blockade showed that mutant receptor levels were no longer significantly different to wild type control receptor levels. Wild type receptors showed a greater effect of caveolin blockade relative to dynamin blockade, but did not reach significance in either case presumably because of the relative stability of these receptors on the cell surface under these conditions. Interestingly, I observed a much greater effect of the dominant negative caveolin-1 on mutant receptor expression than with the dynamin dominant negative expression. I expected that the construct that was used to inhibit dynamin mediated endocytosis (dynamin 1 K44E) would inhibit caveolin mediated endocytosis as another similar construct, dynamin 1 K44A (Le et al., 2002), acts as a dominant negative of all dynamin 2 isoforms (Damke et al., 1994; Henley et al., 1998). (It should be noted that HEK293 cells do not express the brain restricted dynamin 1, but do express various isoforms of dynamin 2 (Henley et al., 1998). Thus, it was a surprise to see that the dominant negative caveolin-1 construct appeared to show an even greater block of endocytosis than dynamin block alone.  134  Figure 4.2. Likely sites of action of the dominant negative endocytosis constructs used in this study. The K44E dynamin mutant blocks both clathrin and caveolin-1 lipid raft dependent endocytosis. The dominant negative, Cav1-DN, blocks caveolin-1 lipid raft endocytosis and may also potentially block caveolin independent lipid raft endocytosis due to overexpression.  lipid raft  non-raft dynamin  Cav-DN  K44E  Endocytosis  Clathrin-coated vesicle  Endosome?  Caveosome  Endosome  Lysosomal degradation  135  While the importance of a threshold level of caveolin and cholesterol is established in the formation of caveolae (Lajoie and Nabi, 2007), determining the precise role of caveolin-1 in non-clathrin dependent endocytosis is still open to debate (Hansen and Nichols, 2009). However, consistent with my own results described here, recent observation of several studies with overexpression of caveolin-1 (Kirkham et al., 2005; Le et al., 2002; Minshall et al., 2000; Sharma et al., 2004) or a dominant negative caveolin-1 (Pelkmans et al., 2001) show an inhibition of raft-dependent endocytosis (Figure 4.2). Interestingly, overexpression of caveolin-1 may also inhibit some noncaveolin lipid raft endocytosis (Le et al., 2002). These observations prompted one group to propose that caveolin-1, through its direct interaction with cholesterol, acts to indirectly regulate raft-dependent endocytosis by changing the endocytic potential of non-caveolar raft domains (Lajoie and Nabi, 2007). Nontheless, specific mechanisms have not been elucidated (Hansen and Nichols, 2009). Thus my results here not only provide evidence for enhanced internalization of mutant GABA-A receptors at the surface, but also suggest that GABA-A receptors may be targeted to rafts were they are subject to caveolin and raft dependent endocytosis.  4.13 Evidence for an interaction between GABA-A receptors and caveolin-1 The effect of dominant negative expression of caveolin-1 relative to dynamin led me to wonder whether a specific interaction may be occurring between GABA-A receptors and caveolin-1. I found that all the α1-6 subunits of GABA-A receptors contain a classic caveolin scaffold binding domain. Coimmunoprecipitation showed that α1 GABA-A receptor subunits could pull-down caveolin-1 and vice versa in HEK293 cells. 136  Preliminary analysis of neuronal rat brain likewise yielded similar results (Bradley and Taghibiglou, unpublished results). Though it was previously thought that neuronal caveolin-1 expression was too low to be of great importance in the trafficking and function of neuronal receptors, recent studies seem to be proving otherwise (Francesconi et al., 2009; Kang et al., 2006b; Kong et al., 2007). For example, the neuronal metabotropic glutamate receptor (mGluR1/5) has been shown to be regulated through the caveolin scaffolding domain (Francesconi et al., 2009) and co-IPs caveolin-1 in HEK293 cells and neuronal lysates. Caveolin-1 modulates the constitutive rate of surface mGluR 1/5 expression by stabilizing the receptor through mGluR1 binding at sites with the TM1 or intracellular loop and possibly TM 6 in mGluR5. Interestingly, mGluR signaling is impaired by caveolin-1 binding suggesting that raft associated mGluRs are functionally silent. In addition, caveolin-1 is upregulated in old age brain tissues of both human and rats and has been linked to an increase in amyloid precursor protein secretion (Kang et al., 2006b). Thus, the finding that caveolin-1 and alpha subunits of the GABA-A receptor are associated, and that they can be stabilized on the PM surface by expression of a dominant negative caveolin-1, opens a new understanding of the trafficking/stability of these receptors in lipid raft and, potentially, at extrasynaptic compartments of neuronal cells.  137  4.14 Mechanisms contributing to the reduction in GABA-A receptor function in the α1(A322D) mutant.  In the initial report from this lab the α1(A322D) mutation caused a reduction in sensitivity to GABA (Cossette et al., 2002). Currently, two different non-exclusive mechanisms have been proposed to explain this effect. The first hypothesis describes a reduction in channel function (Fisher, 2004; Krampfl et al., 2005). Independent analysis of the functional consequences of this mutation with macropatch recordings or single channel recordings showed that the A322D point mutation had an important effect on channel gating kinetics (Fisher, 2004). Thus, in addition to reducing GABA sensitivity, it increased the deactivation rate and decreased the mean channel open time. Channel conductance, however, was unaffected. Similarly enhanced deactivation and resensitization kinetics were determinedly independently by another group and found to be reduced relative to wild type receptors (Krampfl et al., 2005). The second mechanism is based on the hypothesis that there is a reduction in the formation of trafficking competent receptors, which are instead targeted for ERAD (Bradley et al., 2008; Gallagher et al., 2007; Gallagher et al., 2005; Gallagher et al., 2004). I have shown here that degradation of the α1(A322D) subunit and mutant heteromeric receptor occurs and that it is likely to result in ERAD. Consistent with this work, concurrent research presented by Gallagher et al. also showed that ERAD mechanisms were important in the degradation of the mutant subunit and heteromeric receptor (Gallagher et al., 2007; Gallagher et al., 2005). Using endoH treatment, which removes high mannose N-linked glycans of ER resident proteins, the authors also showed  138  drastic reductions in the maturation of the mutant receptor relative to wild type and reduced cell surface expression. In a later paper the authors are able to show directly that the α1A322D mutation results in a fatal misfolding failure some of the time. Using the insertion of a N-glycosylation site between the cytosolic TM3 and TM4 linker (concurrent with knock out of the other glycosylation sites on α1), the authors showed that approximately 33% of the mutant homomeric receptor failed to insert into the lipid bilayer. Failure of lipid insertion was likely to be dependent on a marked change in hydrophobicity. Degradation of the receptor was assessed for changes in Ub dependent proteasomal degradation using lactacystin and was found to be upregulated for the homomeric mutant receptor relative to wild type controls. Assessment of the heterozygous (α1:α1(A322D)) functional expression showed similar EC50 values as the homozygous receptor and further showed a positional dependence in tethered concatamers (Gallagher et al., 2004). Heteromeric receptors in the β-α-β configuration were more stably expressed than β-α- as assessed by western blotting and surface biotinylation and correlated with current reductions that reflected this difference in stability. Based on the ERAD mechanism, it was proposed that the primary deficit in a heterozygous situation is a reduction in the number of wild type receptors (Gallagher et al., 2005). In the work described here, I have shown that the α1(A322D) is a target of ERAD, which is likely caused by misfolding and enhanced calnexin binding (Figure 4.3). The stability of the mutant receptor is unaffected by lowering incubation temperature, but may be partially stabilized by a chemical chaperone, though this affect did not reach significance. In addition, mutant receptors are preferentially degraded by the lysosome.  139  Figure 4.3. A summary of how the α1(A322D) mutation affects the GABA-A receptor. A. Wild type GABA-A receptors are stably expressed on the surface of the cell. There is a low degree of dynamin-dependent endocytosis, which is presumed to be clathrinmediated. B. In contrast, mutant GABA-A receptors are less stably expressed on the cell surface. This is due to endocytosis from the lipid raft (potentially via both dynamindependent and dynamin-independent mechanisms) and probably an increase in the rate of dynamin-dependent, clathrin-mediated endocytosis. In addition, an increase in ERAD results in less mutant receptor reaching the cell surface. Arrow weights and lines indicate proportion of receptors taking this route. Please refer to Figures 1.4 and 4.2 for descriptions of organelles depicted.  140  Figure 4.3. Summary of how the α1(A322D) mutation affects the GABA-A receptor.  Wild type receptor  Mutant receptor  lipid raft  lipid raft  ?  ?  ?  ?  ?  ?  Lysosome  proteasome  Lysosome  Golgi  Endoplasmic Reticulum  proteasome  141  Golgi  Endoplasmic Reticulum  Finally, I have identified another error in the handling of the mutant subunit, or receptor containing the mutant subunit. I found that receptor complexes, which escape ERAD, are available for insertion into the plasma membrane but are less stable than the wild type receptor since they are more readily endocytosed. This endocytosis of the mutant receptor occurs by both dynamin-dependent and caveolin 1-dependent processes. These data suggest that processes that regulate the rate of receptor endocytosis can act as an additional quality control mechanism. Since mutant α1(A322D) containing receptors are clearly inserted into the plasma membrane they will contribute to the total GABA current in heterozygotes. Their shorter half-life in the plasma membrane and altered electrical properties will both contribute to the reduction in GABA current.  4.15 Future Directions A major challenge for the future will be to determine the relative importance of misfolding, enhanced ERAD and altered trafficking of the α1(A322D) mutation for the epilepsy phenotype in an in vivo model. Generation of a knock-in mouse containing the α1(A322D) mutation would be the next appropriate step to be able to directly assess the affects of these mechanistic alterations in a physiologically relevant model. More generally, it would be of interest to determine the extent of intracellular accumulation of mutant GABA-A receptors to determine if aggregates formed in some neurons. In my experiments, expressing this mutant in neurons caused significant cell death. This may be because constitutive expression overloaded the cell’s ability to degrade the protein, but it may also cause more cell death of particular populations of neurons due to cellular stress. It would also be interesting to determine how 142  developmental changes may alter GABA-A receptor function or expression in mutant heterozygous mice and perhaps lead to some answers about why the epilepsy occurs in mid-childhood and not earlier. For example, is the timing of the epileptic phenotype related to changes in expression of the α1 subunit or is it independent of it? In the course of this work on the α1 subunit mutant, I have also discovered that GABA-A receptors are capable of being internalized by a raft endocytosis pathway that is dependent on caveolin-1. Furthermore, I discovered that GABA-A receptors and caveolin-1 are capable of binding one another, possibly through a putative motif for caveolin-1 binding that is conserved in all α subunits of the GABA-A receptor. This suggests that a direct protein-protein interaction may occur in neurons and thus leaves many interesting questions to be examined in future experiments as to how GABA-A receptors might be regulated by caveolin endocytosis. However, my study was done in HEK293 cells and so it would be necessary to firstly test for the association of GABA-A receptors with caveolin-1 in neurons. Next it would be necessary to examine whether there is an effect of manipulating raft-dependent endocytosis on GABA-A receptor surface number in neurons. My preliminary hippocampal co-IPs suggests that the association does exist in neurons, but more replications are required to confirm this result. It is known that GABA is highly susceptible to ligand/modulator induced downregulation and indeed, may be the principal reason for the short usefulness of many mimetic drugs that target GABA-A receptors (Barnes, 1996). It is intriguing to speculate what the purpose of having multiple endocytic routes are for the GABA-A receptor and whether different types of signaling can cause upregulation or downregulation of GABA  143  mediated inhibition through changes in recycling rates or degradations rates. While a relatively new endocytic pathway, caveolin dependent internalization has been shown to be increasingly important in brain function (Gonzalez et al., 2007; Luoma et al., 2008). Interestingly, two new studies, one on the epidermal growth factor (EGF) receptor and the other on transforming growth factor beta (TGFβ) receptor, have demonstrated that the mechanism of receptor internalization is linked to different fates. In the case of the TGFβ receptor, internalization by clathrin and transfer into EEA1 positive endosomes causes signal activation through interaction with resident factors, whereas internalization by the lipid raft caveolar pathway promotes receptor degradation (Di Guglielmo et al., 2003). Similarly, the EGF receptor was found to internalize with low concentration of ligand by clathrin-mediated endocytosis and this pathway promoted recycling of the receptor (Sigismund et al., 2008). At high concentrations, receptor downregulation was mediated by a clathrin-independent internalization method that resulted in receptor degradation. Answering the question of whether caveolin-1 and GABA-A receptors interact directly or indirectly may assist in developing tools to probe this relationship further. GST fusions of caveolin-1 and in vitro binding assays with α subunits can be used to test this hypothesis. To fully characterize the interaction and isolate the true binding domain, truncation deletion and mutational analysis can be undertaken to determine the site of interaction for both proteins. Having isolated the putative interaction domains, it may then be possible to explore the normal function of this interaction under constitutive conditions and in the presence of the ligand. It will also be possible to examine this interaction in pathophysiological conditions such as stroke (Mielke and Wang, 2005) and  144  SE (Mangan et al., 2005; Naylor et al., 2005) where strong internalization of GABAreceptors has been shown to occur shortly after the brain insult. Indeed, from this work on the α1(A322D) and my collaborations in the lab on lipid rafts (Taghibiglou et al., 2009) I began to wonder whether epileptogenesis was initiated or, in part, regulated by GABA-A receptor clathrin-mediated or non-clathrin mediated internalization. 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