Groot-Kormelink, PJ;
Ferrand, S;
Kelley, N;
Bill, A;
Freuler, F;
Imbert, P-E;
Marelli, A;
... Siehler, S; + view all
(2016)
High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor.
PLOS ONE
, 11
(9)
10.1371/journal.pone.0163129.
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Abstract
High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.
| Type: | Article |
|---|---|
| Title: | High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1371/journal.pone.0163129 |
| Publisher version: | http://dx.doi.org/10.1371/journal.pone.0163129 |
| Language: | English |
| Additional information: | © 2016 Groot-Kormelink et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
| Keywords: | Science & Technology, Multidisciplinary Sciences, Science & Technology - Other Topics, ALPHA-SUBUNIT, BINDING-SITE, SELECTIVITY, DISEASE, IDENTIFICATION, PHARMACOLOGY, TRAFFICKING, RESOLUTION, MECHANISM, RESIDUES |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Neuro, Physiology and Pharmacology |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/1516768 |
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