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High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor

Groot Kormelink, Paul, Ferrand, Sandrine, Kelley, Nicholas, Bill, Anke, Freuler, Felix, Imbert, Pierre-Eloi, Marelli, Anthony, Gerwin, Nicole, Sivilotti, Lucia G, Miraglia, Loren, Orth, Anthony, Oakeley, Edward James, Schopfer, Ulrich and Siehler Wagner, Sandra (2016) High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor. PLOS One (Journal), 11 (9). e0163129-e0163129. ISSN 1011-2138


High throughput random mutagenesis is a powerful tool to identify important functional residues in the studied protein in an unbiased way, and to gain insight into its structure-function. The human muscle nicotinic acetylcholine receptor was used to test whether high throughput mutagenesis previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the alpha1 subunit of the channel was generated using error-prone PCR. Novel single molecule real time sequencing was utilized to assess the sequences of the 2816 alpha1 subunit mutants obtained. All alpha1 subunit mutants were co-transfected with wildtype alpha1, beta, delta and epsilon subunits, and functionally studied in an ion flux assay. Mutant hits were extracted based on intact signals for the selective agonist epibatidine, and a loss of either alpha-bungarotoxin or tubocurarine inhibition. Eight alpha1 subunit mutants were identified, and six of them contained mutations of either Y233 or V275. Three mutants with single amino acid changes (Y233N, Y233H, and V275M) were further studied, and demonstrated to enhance the potencies of all five channel agonists tested, suggesting a gain of function and increased ligand sensitivity. This shows that novel single molecule real time sequencing enables to analyze sequences of individual mutants, and that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants.

Item Type: Article
Keywords: muscle nicotinic nAchR, HT mutagenesis, SMRT sequencing, GoF mutants
Date Deposited: 22 Sep 2016 00:45
Last Modified: 22 Sep 2016 00:45


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