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Title: Recombinant glycine receptors : stoichiometry and kinetics
Author: Krashia, P.
ISNI:       0000 0004 2732 7791
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2009
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Glycine receptors (GlyR) are anion-permeable channels that belong to the pentameric ligand-gated ion channel family. Different GlyR subtypes are known. The main synaptic form is thought to be α1β heteropentamers which mediate fast synaptic inhibition in the adult spinal cord and brainstem. Data on recombinant receptors suggest two possible stoichiometries for this subtype, 2α1:3β and 3α1:2β. Evidence for the first comes from experiments on oocytes, whereas a study in mammalian cells favours the latter, raising the possibility that stoichiometry depends on the expression system. Here, we assess the stoichiometry of α1β GlyRs in Xenopus oocytes using two different electrophysiological approaches. The first involves the use of a reporter mutation at the conserved 9΄ position of the pore-lining domain. In other receptors, this mutation shifts agonist sensitivity in proportion to the number of mutated subunits. Recordings from mutant receptors failed to point towards one or the other stoichiometry. The second approach involved single-channel recordings from conductance mutants. This approach was also inconclusive for stoichiometry. However, we provide evidence that oocytes are not a suitable expression system for the study of heteromeric glycine receptors as they are highly prone to contamination by homomers. α2 homomeric GlyRs are predominant early in development and are replaced by α1 subunits in the first postnatal days. We investigated the activation mechanism of these channels in HEK293 cells by maximum likelihood fitting of single-channel data, at a wide range of glycine concentrations. The mechanism we propose suggests that α2 channels can open only when all binding sites are occupied by glycine, and only after the channel undergoes a conformational change ('flip') that links binding to gating. Macroscopic data favour a two binding site model. The scheme can describe adequately macroscopic currents from fast concentration jumps experiments when desensitization is included in the model.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available