title: High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor.
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.
authors.affiliation: Musculoskeletal Disease Area, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Groot-Kormelink
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Ferrand
authors.affiliation: Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Kelley
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America.
authors.forename: Anke
authors.initials: A
authors.lastname: Bill
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Felix
authors.initials: F
authors.lastname: Freuler
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Pierre-Eloi
authors.initials: PE
authors.lastname: Imbert
authors.affiliation: Genomics Institute of the Novartis Research Foundation, Novartis Institutes for BioMedical Research, San Diego, California, United States of America.
authors.forename: Anthony
authors.initials: A
authors.lastname: Marelli
authors.affiliation: Musculoskeletal Disease Area, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Nicole
authors.initials: N
authors.lastname: Gerwin
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom.
authors.forename: Lucia G
authors.initials: LG
authors.lastname: Sivilotti
authors.affiliation: Genomics Institute of the Novartis Research Foundation, Novartis Institutes for BioMedical Research, San Diego, California, United States of America.
authors.forename: Loren
authors.initials: L
authors.lastname: Miraglia
authors.affiliation: Genomics Institute of the Novartis Research Foundation, Novartis Institutes for BioMedical Research, San Diego, California, United States of America.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Orth
authors.affiliation: Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Oakeley
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schopfer
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Siehler
mesh_terms: D000595:Amino Acid Sequence; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002038:Bungarotoxins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D009132:Muscles / Q000378:metabolism*; D016296:Mutagenesis; D009154:Mutation; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014403:Tubocurarine / Q000494:pharmacology
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D002038:Bungarotoxins; C523021:CHRNA1 protein, human; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011725:Pyridines; D011978:Receptors, Nicotinic; C082748:epibatidine; D002118:Calcium; D014403:Tubocurarine