Structural biology has become an important part of researching various diseases and drug development. In this thesis, I provide details on how I worked with approaches to structural characterization of a heteromeric GABA(A)R. These pentameric ligand gated ion channels take part in regulating inhibition of action potentials in nerve cells by allowing the passage of Cl- ions when bound by gamma-aminobutyric acid (GABA). They are formed by the assembly of five subunits which can be of various different types, denoted by greek letters and a number. Much is still unknown about how GABA and several other ligands bind to these ion channels and how that impacts function. Obtaining a structure of these proteins can aid in closing those knowledge gaps. It is reasonable to screen the proteins you have before you study their structures by Cryo-EM in order to get the best result, a methodology for which is described here. I have followed this methodology to screen two heteromeric GABA$_A$R that we wish to determine the structure of, alpha 5 beta 3 and rho 1 gamma 2. Neither of the combinations of genes we used to express these proteins proved to produce the desired fully assembled heteromeric protein. In the case of alpha 5 beta 3, we only witnessed building blocks, with no fully assembled channels. In rho 1 gamma 2, we instead only witnessed fully formed homomers of the rho 1 subunit. These findings then exclude the gene constructs used from further structural study, and the methodology described will inform the next steps to be taken. / Strukturbiologi har blivit en viktig del av forskningen kring många sjukdomar samt utveckling av läkemedel. I denna uppsats delger jag hur jag arbetat med metoder för strukturell karakterisering av en heteromerisk GABA(A)R. Dessa pentameriska ligandstyrda jonkanaler deltar i regleringen av hämning av aktionspotentialer i nervceller genom att tillåta passagen av Cl- joner när gamma-aminosmörsyra (GABA) binder. Dessa består av fem subenheter som kan vara en av flera olika typer, vilka anges med en grekisk bokstav och en siffra. Mycket om hur GABA och andra ligander binder till dessa jonkanaler och hur det påverkar dess funktion är fortfarande okänt. Att hitta en struktur av dessa proteiner kan hjälpa oss att stänga kunskapsgapen. Det är klokt att undersöka om genen man ska använda för att uttrycka det sökta proteinet ger det man söker innan man sen börjar studera strukturen. Jag har beskrivit en metodologi för detta och följt den för två heteromeriska proteiner, alpha 5 beta 3 och rho 1 gamma 2. Ingen av kombinationerna av gener vi använt för att uttrycka dessa proteiner har producerat de sökta, fullt ihoppbyggda proteinerna. I fallet för alpha 5 \beta 3 så ser vi endast byggstenar och inga kompletta proteiner, och för rho 1 gamma 2 så ser vi endast homomeriska proteiner av rho 1. Dessa slutsatser exkluderar de genkonstruktioner vi använt från vidare strukturella studier, och stegen som bör tas härnäst beskrivs av den använda metodologin.

Past research has demonstrated that the motivational effects of opiates causes a change in ventral tegmental area (VTA) γ-amino butyric acid (GABA) subtype A receptor [GABA(A)R] complexes in opiate-dependent animals, which switch from a GABA-induced hyperpolarization of VTA GABA neurons to a GABA-induced depolarization. Previously shown in naïve animals, superfusion of ethanol (IC50 = 30 mM) and the GABA(A)R agonist muscimol (IC50 = 100 nM) decreased VTA GABA neuron firing rate in a dose-dependent manner. The aim of this study was to evaluate VTA GABA neuron excitability, GABA synaptic transmission to VTA GABA neurons, and a potential switch in GABA(A)R functionality produced by alcohol dependence. To accomplish these studies, we used standard whole-cell, perforated patch, and attached-cell mode electrophysiological techniques to evaluate chronic ethanol effects on VTA GABA neurons in CD-1 GAD GFP mice, which enable the visual identification of GABA neurons in the slice preparation. In order to more conclusively demonstrate synaptic plasticity in VTA neurons associated with alcohol dependence, three studies were proposed to elucidate the mechanism underlying the switch in GABA synaptic function with dependence. First, we evaluated the effects of withdrawal from chronic ethanol exposure on muscimol-induced inhibition of VTA GABA neuron firing rate. Second, we evaluated the effects of withdrawal from chronic ethanol exposure on GABA(A)R-mediated synaptic responses in VTA GABA neurons by looking at eIPSCs, and corresponding changes in VTA DA neuron firing rate. Third, we evaluated chloride reversal potentials in VTA GABA neurons using perforated patch recordings in VTA GABA neurons.Through these studies, we found that there was less sensitivity to muscimol in animals treated with ethanol versus air-exposed controls. However, it is yet to be shown more conclusively if VTA GABA neurons undergo a switch in GABA(A)R function with chronic ethanol.