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

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

The Startle Disease Mutation E103K Impairs Activation of Human Homomeric α1 Glycine Receptors by Disrupting an Intersubunit Salt Bridge across the Agonist Binding Site

Glycine receptors (GlyR) belong to the pentameric ligand-gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface. Homology modeling shows that the side chain of Glu-103 is close to that of Arg-131, in loop E of the complementary side of the binding site, and may form a salt bridge at the back of the binding site, constraining its size. We investigated this hypothesis in recombinant human α1 GlyR by site-directed mutagenesis and functional measurements of agonist efficacy and potency by whole cell patch clamp and single channel recording. Despite its position near the binding site, E103K causes hyperekplexia by impairing the efficacy of glycine, its ability to gate the channel once bound, which is very high in wild type GlyR. Mutating Glu-103 and Arg-131 caused various degrees of loss-of-function in the action of glycine, whereas mutations in Arg-131 enhanced the efficacy of the slightly bigger partial agonist sarcosine (N-methylglycine). The effects of the single charge-swapping mutations of these two residues were largely rescued in the double mutant, supporting the possibility that they interact via a salt bridge that normally constrains the efficacy of larger agonist molecules

Three Rounds of Read Correction Significantly Improve Eukaryotic Protein Detection in ONT Reads

Background: Eukaryotes’ whole-genome sequencing is crucial for species identification, gene detection, and protein annotation. Oxford Nanopore Technology (ONT) is an affordable and rapid platform for sequencing eukaryotes; however, the relatively higher error rates require computational and bioinformatic efforts to produce more accurate genome assemblies. Here, we evaluated the effect of read correction tools on eukaryote genome completeness, gene detection and protein annotation. Methods: Reads generated by ONT of four eukaryotes, C. albicans, C. gattii, S. cerevisiae, and P. falciparum, were assembled using minimap2 and underwent three rounds of read correction using flye, medaka and racon. The generates consensus FASTA files were compared for total length (bp), genome completeness, gene detection, and protein-annotation by QUAST, BUSCO, BRAKER1 and InterProScan, respectively. Results: Genome completeness was dependent on the assembly method rather than on the read correction tool; however, medaka performed better than flye and racon. Racon significantly performed better than flye and medaka in gene detection, while both racon and medaka significantly performed better than flye in protein-annotation. Conclusion: We show that three rounds of read correction significantly affect gene detection and protein annotation, which are dependent on assembly quality in preference to assembly completeness