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Exploring the structure function relation of the glycine receptor with hyperekplexia mutations

Safar, Fatemah; (2018) Exploring the structure function relation of the glycine receptor with hyperekplexia mutations. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The glycine receptor (GlyR) is a Cys-loop ligand-gated anion channel that mediates fast synaptic inhibition in brain and spinal cord. Heritable malfunction of glycinergic transmission in man causes hyperekplexia, a neuromotor disorder characterised by exaggerated startle responses to normal sensory stimuli. Many mutations responsible for the disease are found in GlyR subunits, where they highlight residues essential for channel activation. I evaluated the effects of four human hyperekplexia 1 subunit mutations located in different parts of the GlyR including the extracellular domain (ECD), transmembrane domain (TM1) and transmembrane domain (TM2). Human α1 and 1 GlyR bearing the E103K, S231N, Q266H or S267N mutations in α1 were expressed in HEK293 cells. Glycine concentrationresponse curves obtained by whole-cell patch-clamp recordings confirmed previous reports (Bode & Lynch, 2014) that these mutations decrease the channel sensitivity to glycine, increasing its EC50. To understand the mechanism of action of these mutations, I performed also single-channel recordings (cell-attached, pipette potential +100 mV) at saturating glycine concentrations. This allowed measurement of the channel maximum open probability (Popen = cluster open time / total cluster time). The mutations tested decreased the GlyR maximum Popen to 0.37 – 0.67, cf. the wild-type value of 0.98. This reduction in maximum Popen was clear, despite the presence of distinct gating modes (stretches of activations with different Popen) in mutant receptors. These data suggest that the human hyperekplexia mutations tested here increase glycine EC50 by reducing gating efficacy. To determine whether the function of the mutant GlyRs can be rescued, the intravenous anasthetic, propofol was used. Propofol (50 μM) was found to enhance responses to submaximal glycine concentrations in all heteromeric receptors (by 2.71 - 5.19-fold). However, the impaired maximum response of mutant receptors was increased by propofol only for the S231N mutant GlyR. Residues in the ECD are likely to be vital for agonist recognition and might have influence on channel gating. This was the case with the hyperkplexia α1 E103K GlyR mutation. In order to explain that, I investigated the role of residues at the back of the binding site, in loops A and E, E103 and R131, respectively, and established that they interact, probably by forming an intersubunit salt-bridge that is crucial for channel gating of the glycine receptor. The interruption of this interaction might explain the reason behind the effect of the E103K hyperekplexia mutation.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Exploring the structure function relation of the glycine receptor with hyperekplexia mutations
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
URI: http://discovery.ucl.ac.uk/id/eprint/10050853
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