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Title: The activation mechanism of the α1β heteromeric glycine receptor
Author: Burzomato, Valeria
ISNI:       0000 0001 3511 7944
Awarding Body: University College London
Current Institution: University College London (University of London)
Date of Award: 2005
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α1β glycine receptors (GlyR) mediate fast inhibition in the adult mammalian brainstem and spinal cord: the aim of this work is to understand the activation mechanism of this receptor. Firstly we developed a transfection protocol for expressing α1β GlyRs with minimal contamination by α1 homomers. This was achieved by using an extremely high ratio (40:1) in the amount of β to α subunit cDNA transfected in HEK 293 cells. We then investigated the receptor stoichiometry, using a hydrophilic mutation in the conserved 9' position of the pore-lining region of the receptor. In the nicotinic superfamily the decrease in EC50 produced by this type of mutation is proportional to the number of mutant subunit incorporated. EC50 data supported a 3α:2β stoichiometry. This information is useful in choosing putative kinetic schemes. The activation mechanism was investigated by fitting putative mechanisms to singlechannel recordings obtained at four glycine concentrations (10-1000 μM). We identified several features of the mechanism essential for adequate fits: three agonist binding sites, the presence of both partially- and fully-liganded openings a strong cooperative interaction between successive binding steps, leading to a facilitation of binding as more agonist molecules bind. The hypothesis that binding sites have different affinities to start with was not supported by our fits. Two types of models were found to provide adequate description of the data: in the first, binding cooperativity appears to be due to interaction between the binding sites (each site senses the state of ligation of the others). In the second, agonist binding stabilises a higher affinity form of the receptor that is produced by a conformational change ('flip') preceding the channel opening. Although the two schemes describe our data equally well, the latter has fewer free parameters and offers an elegant and plausible explanation for the affinity increase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available