Fluorescent labelling of glycine receptors with the unnatural amino acid ANAP
-
1
The University of Queensland, Queensland Brain Institute, Australia
-
2
Monash University, Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Australia
-
3
The University of Queensland, School of Biomedical Sciences, Australia
We are interested in understanding how glycine receptor (GlyR) chloride ion channels move between closed, open and desensitised states in real time. This is traditionally achieved with voltage-clamp fluorometry (VCF), which involves labelling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Sulfhydryl-tagged rhodamine dyes have been used for this purpose, although drawbacks include bulky molecular size and limited labelling sites. A recently developed small fluorescent unnatural amino acid, ANAP, could be directly incorporated into any part of the receptor site-specifically using the nonsense suppression approach, although this has never been successfully done in any Cys-loop receptor. Here we describe the novel incorporation of ANAP into human a1GlyRs to study the functional states, structural rearrangements and receptor interaction during activation by different agonists using VCF. We first inject the plasmid of an orthogonal ANAP-specific tRNACUAaminoacyl-tRNA synthetase pair into the nucleus of Xenopus laevis oocytes, followed by the cytoplasmic injection of ANAP and a1GlyR mRNA containing the amber stop-codon mutation at the site of interest. The successful incorporation of ANAP was confirmed by the expression of glycine-activated currents and by confocal microscopy showing the distinct emission spectrum of ANAP compared to uninjected oocytes. We showed that glycine, ß-alanine and taurine induced similar current magnitudes but distinct fluorescence changes in loops 2, 7 and 9, indicating that these sites are involved in isomerising the receptors into a pre-open flip state. These results provide information about the structural basis of partial agonism in GlyRs and represent an evolved VCF technique.
Keywords:
in vivo,
Amino acid,
glycine receptor,
oocyte,
Fluorescent labelling
Conference:
14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016.
Presentation Type:
O02: Postgraduate Travel Awardees Oral Session 2
Topic:
14th Meeting of the Asian-Pacific Society for Neurochemistry
Citation:
Soh
MS,
Estrada-Mondragon
A,
Durisic
N and
Lynch
JW
(2016). Fluorescent labelling of glycine receptors with the unnatural amino acid ANAP.
Conference Abstract:
14th Meeting of the Asian-Pacific Society for Neurochemistry.
doi: 10.3389/conf.fncel.2016.36.00085
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
04 Aug 2016;
Published Online:
11 Aug 2016.
*
Correspondence:
Prof. Joseph W Lynch, The University of Queensland, Queensland Brain Institute, St Lucia, Queensland, Australia, j.lynch@uq.edu.au