Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel.
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. 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.
Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden.
Stephanie A
0000-0003-3224-4547
SA
Heusser
Swedish e-Science Research Center, KTH Royal Institute of Technology, Stockholm, Sweden.
Özge
0000-0001-8354-0253
Ö
Yoluk
Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden.
Göran
0000-0001-8816-3186
G
Klement
Department of Chemistry, Skidmore College, Saratoga Springs, NY, USA.
Erika A
0000-0003-1011-6536
EA
Riederer
Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden.|Swedish e-Science Research Center, KTH Royal Institute of Technology, Stockholm, Sweden.
Erik
0000-0002-2734-2794
E
Lindahl
Department of Chemistry, Skidmore College, Saratoga Springs, NY, USA.
Rebecca J
0000-0003-2049-3378
RJ
Howard
D000818:Animals; D018118:Chloride Channels / Q000378:metabolism*; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D058446:Ligand-Gated Ion Channels / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D018377:Neurotransmitter Agents / Q000378:metabolism*; D014981:Xenopus
D018118:Chloride Channels; D058446:Ligand-Gated Ion Channels; D018377:Neurotransmitter Agents; C109777:glutamate-gated chloride channels; D018698:Glutamic Acid
GluCl; Ion channel; anesthetic; cys-loop receptor; pLGIC