Roles for loop 2 residues of alpha1 glycine receptors in agonist activation.
The present study tested the hypothesis that several residues in Loop 2 of alpha1 glycine receptors (GlyRs) play important roles in mediating the transduction of agonist activation to channel gating. This was accomplished by investigating the effect of cysteine point mutations at positions 50-60 on glycine responses in alpha1GlyRs using two-electrode voltage clamp of Xenopus oocytes. Cysteine substitutions produced position-specific changes in glycine sensitivity that were consistent with a beta-turn structure of Loop 2, with odd-numbered residues in the beta-turn interacting with other agonist-activation elements at the interface between extracellular and transmembrane domains. We also tested the hypothesis that the charge at position 53 is important for agonist activation by measuring the glycine response of wild type (WT) and E53C GlyRs exposed to methanethiosulfonate reagents. As earlier, E53C GlyRs have a significantly higher EC(50) than WT GlyRs. Exposing E53C GlyRs to the negatively charged 2-sulfonatoethyl methanethiosulfonate, but not neutral 2-hydroxyethyl methanethiosulfonate, positively charged 2-aminoethyl methanethiosulfonate, or 2-trimethylammonioethyl methanethiosulfonate, decreased the glycine EC(50) to resemble WT GlyR responses. Exposure to these reagents did not significantly alter the glycine EC(50) for WT GlyRs. The latter findings suggest that the negative charge at position 53 is important for activation of GlyRs through its interaction with positive charge(s) in other neighboring agonist activation elements. Collectively, the findings provide the basis for a refined molecular model of alpha1GlyRs based on the recent x-ray structure of a prokaryotic pentameric ligand-gated ion channel and offer insight into the structure-function relationships in GlyRs and possibly other ligand-gated ion channels.
Crawford
Daniel K
DK
Alcohol and Brain Research Laboratory, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089; Neuroscience Graduate Program, University of Southern California, Los Angeles, California 90089.
Perkins
Daya I
DI
Alcohol and Brain Research Laboratory, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089.
Trudell
James R
JR
Department of Anesthesia and Beckman Program for Molecular and Genetic Medicine, Stanford School of Medicine, Stanford, California 94305.
Bertaccini
Edward J
EJ
Department of Anesthesia and Beckman Program for Molecular and Genetic Medicine, Stanford School of Medicine, Stanford, California 94305; Department of Anesthesia, Palo Alto Veterans Affairs Health Care System, Palo Alto, California 94304.
Davies
Daryl L
DL
Alcohol and Brain Research Laboratory, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089. Electronic address: ddavies@usc.edu.
Alkana
Ronald L
RL
Alcohol and Brain Research Laboratory, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089; Neuroscience Graduate Program, University of Southern California, Los Angeles, California 90089.
D019943:Amino Acid Substitution; D000818:Animals; D005260:Female; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008958:Models, Molecular; D009865:Oocytes / Q000166:cytology; D017354:Point Mutation; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D018009:Receptors, Glycine / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D018009:Receptors, Glycine