Novel Insights Into the Activation of Glycine Receptors

Stephan Alexander Pless (2008). Novel Insights Into the Activation of Glycine Receptors PhD Thesis, School of Biomedical Sciences, The University of Queensland.

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Author Stephan Alexander Pless
Thesis Title Novel Insights Into the Activation of Glycine Receptors
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
Publication date 2008-08-01
Thesis type PhD Thesis
Supervisor Associate Professor Joseph Lynch
Professor David Adams
Total pages 236
Subjects 320000 Medical and Health Sciences
Formatted abstract
The glycine receptor is a pentameric chloride-selective ion channel activated by glycine. It is a member of the Cys-loop receptor family and mediates fast inhibitory neurotransmission in the nervous system. The goal of my project was to provide new insights into how the glycine receptor (GlyR) opens and closes. In particular, aspects of binding, gating and subunit cooperativity were examined during the course of this project. The first part of the project sought to establish a possible cation-π interaction in the GlyR binding site. Such interactions between an aromatic amino acid side chain of the receptor and the amino group of its endogenous ligand have been shown to contribute to ligand binding in other Cys-loop receptor family members. Here we used the nonsense suppression methodology for incorporation of unnatural amino acids to probe possible cation-π interactions in the GlyR binding site. We find a strong correlation between the EC50 and the cation-π binding ability of the fluorinated phenylalanine derivatives incorporated at position 159. We conclude that F159 forms a cation-π interaction with the glycine amino group. We found no such correlation for other aromatic side chains in the binding site (i.e., F63, Y161 and F207) and were thus able to rule out cation-π interactions at these positions. The data thus provides important information on the exact ligand orientation in the GlyR binding site.
We next sought to examine conformational rearrangements in the ligand-binding domain induced by binding of agonists and antagonists. To address this question, we used voltageclamp fluorometry (VCF). This technique allows us to simultaneously follow conformational changes at the channel gate (through electrophysiology) and a domain of interest (through the fluorescence emission of an environmentally sensitive fluorophore tethered to a cysteine residue in the domain of interest). We mutated various residues in different loops and β-sheets of the ligand-binding domain and found a clear correlation between receptor activation and fluorescence readout in a residue in loop E (L127C). All other positions tested here seem to undergo conformational rearrangements only at high glycine concentrations. This raises the questions of whether large parts of the GlyR ligand-binding domain are mainly involved in locking the ligand into the binding pocket rather than actively contributing to channel gating.
We then sought to examine conformational rearrangements at the extracellular end of the pore-lining M2 domain. Models describing the structural changes mediating Cys-loop receptor activation generally give little attention to the possibility that different agonists may induce distinct structural rearrangements in the pore-lining M2 domain. We investigated this question by comparing the effects of different ligands on the conformation of the external portion M2. Conformational flexibility was assessed by using VCF at the R271C or the L274C residue. During glycine activation, the fluorescence increase correlated well with channel activation at R271C. In contrast, ivermectin activated the receptors without producing a fluorescence change. Taurine and β-alanine, however, induced no currents but did produce a fluorescence change. Glycine and taurine (or β-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to L274C. Thus, results from two separate labeled residues support the conclusion that the GlyR M2 domain responds with distinct conformational changes to activation by different agonists.
To address the question of possible inter-subunit cooperativity and whether GlyRs are activated by a concerted or a sequential mechanism, we monitored the M2 domain conformation by using VCF  at the R271C residue. By varying subunit expression levels, we generated heteromeric GlyRs comprising WT plus R271C subunits or WT plus R65K,T204A,R271C subunits, the latter subunit being incapable of glycine binding. In both
receptors, fluorescence signals were observed only when the glycine concentration was high enough to almost fully activate the receptor. Two conclusions can be drawn from this observation. First, because the labeled glycine-free subunit can be induced to change conformation to the activated state by glycine binding to neighbouring WT subunits, it provides evidence for strong subunit cooperativity. However, because the fluorescent label on glycine-free subunits does not move in concert with those attached to glycine-bound subunits, GlyR activation can be described at least partly as a sequential process.
Taken together, the use of innovative techniques has provided novel insights into the activation mechanisms of GlyRs (and perhaps of other Cys-loop receptor family members), particularly agonist binding, structural rearrangements during gating and subunit cooperativity
Keyword Glycine receptor, binding, gating, subunit cooperativity, unnatural amino acids,

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Created: Wed, 19 Nov 2008, 03:35:13 EST by Catherine Kelley on behalf of Library - Information Access Service