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Reconstitution and in-vitro activation of the prokaryotic pentameric ligand-gated ion channel ELIC

Shafaat, Oliver S. and Rusinova, Radda and Winkler, Jay R. and Andersen, Olaf S. and Gray, Harry B. and Dougherty, Dennis A. (2015) Reconstitution and in-vitro activation of the prokaryotic pentameric ligand-gated ion channel ELIC. Abstracts of Papers of the American Chemical Society, 249 . BIOL-246. ISSN 0065-7727.

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Pentameric ligand- gated ion channels are a large class of ion channels that aid in signal transduction between cells. Eukaryotic members of this family include the cation- selective nicotinic acetylcholine receptor (nAChR) and the anion- selective γ- aminobutyric acid (GABA) receptor. Signal transduction and activation follow two processes: first, ligand binds to the channel; and second, a conformational change occurs that leads to channel opening. The ligand binding interactions have been elucidated in numerous studies. However, the subsequent conformational changes that lead to channel opening remain elusive. Recently, prokaryotic members of this ion channel family were isolated, and high- resoln. structures obtained. An activation mechanism was proposed based on the assignment of these structures to putative 'open' and 'closed' states. Activation, however, is dynamic; initiated with ligand binding, and followed by conformational changes that result in pore opening and ion flux through the channel. The sequence of steps that connect the initial ligand binding to channel opening remain poorly understood. Our goal is to explore these steps using dynamic exptl. techniques. Continuous- flow ultrafast (μs) mixing permits delivery of ligand to channels reconstituted into lipid vesicles of defined compn. Combined with time- resolved energy transfer, this approach allows for high- resoln. monitoring of the dynamics of activation. To that end, the prokaryotic ligand- gated ion channel ELIC has been reconstituted into large unilamellar lipid vesicles and activation was monitored using a stopped- flow fluorescence- quench assay. Activation characteristics of the reconstituted ELIC, and developments toward monitoring the early activation steps in the ultrafast mixer, will be discussed.

Item Type:Article
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URLURL TypeDescription Website
Winkler, Jay R.0000-0002-4453-9716
Gray, Harry B.0000-0002-7937-7876
Dougherty, Dennis A.0000-0003-1464-2461
Additional Information:© 2015 American Chemical Society.
Record Number:CaltechAUTHORS:20150423-131529413
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:56921
Deposited By: Tony Diaz
Deposited On:23 Apr 2015 21:26
Last Modified:22 Nov 2019 09:58

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