TY - JOUR
T1 - Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel
AU - Heusser, Stephanie A
AU - Yoluk, Özge
AU - Klement, Göran
AU - Riederer, Erika A
AU - Lindahl, Erik
AU - Howard, Rebecca J
N1 - © 2016 International Society for Neurochemistry.
PY - 2016/7/1
Y1 - 2016/7/1
N2 - Abstract: The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. (Figure presented.) In this study, we elucidate the validity of a modified glutamate-gated chloride channel (GluClcryst) as a structurally accessible model for GABAA receptors. In contrast to native-like controls, GluClcryst exhibits classical activation by its neurotransmitter ligand L-glutamate. The modified channel is also sensitive to allosteric modulators associated with human GABAA receptors, and to site-directed mutations predicted to alter channel opening.
AB - Abstract: The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. (Figure presented.) In this study, we elucidate the validity of a modified glutamate-gated chloride channel (GluClcryst) as a structurally accessible model for GABAA receptors. In contrast to native-like controls, GluClcryst exhibits classical activation by its neurotransmitter ligand L-glutamate. The modified channel is also sensitive to allosteric modulators associated with human GABAA receptors, and to site-directed mutations predicted to alter channel opening.
KW - Animals
KW - Chloride Channels/metabolism
KW - Female
KW - Glutamic Acid/metabolism
KW - Humans
KW - Ion Channel Gating
KW - Ligand-Gated Ion Channels/metabolism
KW - Mutagenesis, Site-Directed/methods
KW - Neurotransmitter Agents/metabolism
KW - Xenopus
U2 - 10.1111/jnc.13644
DO - 10.1111/jnc.13644
M3 - Journal article
C2 - 27102368
SN - 0022-3042
VL - 138
SP - 243
EP - 253
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
IS - 2
ER -