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.
From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Fatemah
F
Safar
From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Elliot
E
Hurdiss
From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Marios
M
Erotocritou
From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Timo
T
Greiner
From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Remigijus
0000-0002-6604-0828
R
Lape
the School of Physiology and Pharmacology, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom, and.
Mark W
MW
Irvine
the School of Physiology and Pharmacology, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom, and.
Guangyu
G
Fang
the School of Physiology and Pharmacology, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom, and.
David
D
Jane
the Department of Biochemistry, Structural Bioinformatics and Computational Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.|the Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China.
Rilei
R
Yu
the Department of Biochemistry, Structural Bioinformatics and Computational Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Marc A
MA
Dämgen
the Department of Biochemistry, Structural Bioinformatics and Computational Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Philip C
0000-0001-5100-8836
PC
Biggin
From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom, l.sivilotti@ucl.ac.uk.
Lucia G
0000-0003-2510-8424
LG
Sivilotti
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D005998:Glycine / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000071017:Hyperekplexia / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D018009:Receptors, Glycine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012521:Sarcosine / Q000378:metabolism; D016415:Sequence Alignment
D018009:Receptors, Glycine; D005998:Glycine; D012521:Sarcosine
agonists; efficacy; glycine receptor; homology modeling; ion channel; mutagenesis; patch clamp; potency; site-directed mutagenesis