Dataset Information

Activation and desensitization of the olfactory cAMP-gated transduction channel: identification of functional modules.

ABSTRACT: Olfactory receptor neurons respond to odor stimulation with a receptor potential that results from the successive activation of cyclic AMP (cAMP)-gated, Ca(2+)-permeable channels and Ca(2+)-activated chloride channels. The cAMP-gated channels open at micromolar concentrations of their ligand and are subject to a Ca(2+)-dependent feedback inhibition by calmodulin. Attempts to understand the operation of these channels have been hampered by the fact that the channel protein is composed of three different subunits, CNGA2, CNGA4, and CNGB1b. Here, we explore the individual role that each subunit plays in the gating process. Using site-directed mutagenesis and patch clamp analysis, we identify three functional modules that govern channel operation: a module that opens the channel, a module that stabilizes the open state at low cAMP concentrations, and a module that mediates rapid Ca(2+)-dependent feedback inhibition. Each subunit could be assigned to one of these functions that, together, define the gating logic of the olfactory transduction channel.

SUBMITTER: Waldeck C

PROVIDER: S-EPMC2768803 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Activation and desensitization of the olfactory cAMP-gated transduction channel: identification of functional modules.

Waldeck Clemens C Vocke Kerstin K Ungerer Nicole N Frings Stephan S Möhrlen Frank F

The Journal of general physiology 20091012 5

Olfactory receptor neurons respond to odor stimulation with a receptor potential that results from the successive activation of cyclic AMP (cAMP)-gated, Ca(2+)-permeable channels and Ca(2+)-activated chloride channels. The cAMP-gated channels open at micromolar concentrations of their ligand and are subject to a Ca(2+)-dependent feedback inhibition by calmodulin. Attempts to understand the operation of these channels have been hampered by the fact that the channel protein is composed of three di ...[more]

PMID: 19822638

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Project description: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.

Dataset Information

Activation and desensitization of the olfactory cAMP-gated transduction channel: identification of functional modules.

Publications

Activation and desensitization of the olfactory cAMP-gated transduction channel: identification of functional modules.

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