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The Startle Disease Mutation E103K Impairs Activation of Human Homomeric ?1 Glycine Receptors by Disrupting an Intersubunit Salt Bridge across the Agonist Binding Site.

ABSTRACT: 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 ?1 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.

SUBMITTER: Safar F

PROVIDER: S-EPMC5377815 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Safar Fatemah F Hurdiss Elliot E Erotocritou Marios M Greiner Timo T Lape Remigijus R Irvine Mark W MW Fang Guangyu G Jane David D Yu Rilei R Dämgen Marc A MA Biggin Philip C PC Sivilotti Lucia G LG

The Journal of biological chemistry 20170207 12

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 subu ...[more]

PMID: 28174298

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Project description:The functional properties of rat homomeric alpha 1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat alpha1 subunit cDNA. Whole-cell dose-response curves gave EC(50) estimates between 30 and 120 microM and a Hill slope of approximately 3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 microM) to outside-out patches. Single channel conductances were mostly 60-90 pS, but smaller conductances of approximately 40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency.

Dataset Information

The Startle Disease Mutation E103K Impairs Activation of Human Homomeric ?1 Glycine Receptors by Disrupting an Intersubunit Salt Bridge across the Agonist Binding Site.

Publications

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

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