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.

Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast neurotransmission in the central nervous system (CNS). GlyRs are pentameric channels, organized quasi-symmetrically around an ion-conducting pore. Opening of the integral ion pore depends on ligand binding and transduction of this binding signal to the channel gate. Research presented in this dissertation describes a number of critical electrostatic interactions that play a role in conserving the closed-state stability of the receptor in the absence of ligand, ensuring that receptor activation occurs only upon neurotransmitter binding. These amino acids, aspartic acid at position 97 (D97), lysine 116 (K116), arginine 119 (R119) and arginine R131 (R131) are charged residues that interact with one another through electrostatic attraction. When D97 is replaced with any other amino acid this destabilizes the closed state of the channel and causes spontaneous GlyR channel opening. I show that restoration of this electrostatic interaction in GlyR bearing double mutations in which the charges are swapped (D97R/R119E and D97R/R131D) markedly decreases this spontaneous current. In addition, I investigate how these residues that interact at the interfaces between receptor subunits affect the efficacies of GlyR partial agonists. My work shows that the partial agonist taurine is converted into a full agonist at both D97R and R131D receptors. Furthermore, I analyzed the structure of the more extracellular part of the transmembrane (TM) 2 segment that lines the ion channel pore, showing that it is unlikely that this fragment (stretching from T13’ to S18’) is constrained in a true alpha helical conformation. From this work, using disulfide trapping and whole cell electrophysiology, I conclude that a significant level of flexibility characterizes this part of the TM2 domain. This segment includes residue S267, previously shown to be significant for alcohol and anesthetic actions, as well as residue Q266 that, when mutated, produces a hyperekplexia-like phenotype. The range of movement of residues in this region may therefore play an important role not only in channel gating but also in how modulators of GlyR function exert their actions. / text