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.

Fig 1. Principles of SMRT sequencing and other NGS methods.

Schematic drawing of SMRT sequencing (PacBio RSII) and other NGS techniques such as an Illumina 454 reader. (A) The complete region covering the full-length coding sequence of the α1 subunit in each mutant cDNA (1371 bp) was sequenced using SMRT sequencing, whereas only up to 300 bp stretches would have been read by an Illumina reader. (B) SMRT sequencing can link each of three fictive mutations (red, blue, green) to the individual mutant, whereas this information is lost with Illumina sequencing due to the amplification of short stretches only.

Fig 2. Error-prone PCR mediated mutagenesis of the nAChR α1 subunit.

(A) Number and distribution of mutations per clone after error-prone PCR mediated mutagenesis of the nAChR α1 subunit. Black bars represent the number of mutations after standard full-length Sanger sequencing of 48 mutants. Grey bars represent the sequenced mutational spectrum at a mutation rate of 1.15/kb using HT-MSP [1]. (B) Expected number and distribution of nucleotide (grey) or amino acid (black) changes in the whole library predicted by HT-MSP with a mutation rate of 1.15/kb. (C, D) Comparison of the distribution of nucleotide (C) and amino acid (D) changes as predicted by HT-MSP (grey) and as measured by SMRT-sequencing (black) for the total library.

Fig 3. Mutational bias analyses of the α1 subunit mutant library.

(A) Number of mutations for each of the 1371 nucleotides of the α1 subunit sequence according to SMRT sequencing data. (B) Frequency of amino acid (AA) changes found in all 2816 α1 subunit mutants. The left column shows the wildtype amino acid, whereas the top letters show the mutated amino acid with (*) representing a mutation into a stop codon. The absolute number (n°) of each type of amino acid within the α1 protein, and the absolute number of mutations for this amino acid are depicted on the right.

Fig 4. Illustration of the functional α1 mutant screen.

(A) The muscle nAChR contains two α1 subunits that form a pentamer together with co-transfected β1δε subunits. Ca²⁺ flux through channels containing wildtype or mutant α1 subunits was measured in a Na⁺-free buffer using the Ca²⁺-dye Fluo4. Typical raw Ca²⁺ signal traces over time are presented for (B) the buffer control, (C) the agonist (epibatidine) control, (D) the blocker tubocurarine, or (E) the blocker α-BTX (both added 30 min before adding the agonist, the injection of which is indicated by an arrow). Agonist-stimulated Ca²⁺ signals of wildtype (Wt) muscle nAChR (black lines) are inhibited by tubocurarine or α-BTX. A non-functional mutant (green lines) is inactive in all conditions, and does not show any Ca²⁺ signals. An α-BTX resistant mutant (blue lines) or tubocurarine resistant mutant (red lines) shows agonist signals in the absence and presence of the respective blocker.

Fig 5. α1-Y233 and α1-Y275 mutants enhance potencies of channel agonists.

(A-E) Concentration response curves of five agonists measured in the Ca²⁺ flux assay for transiently expressed wildtype and mutant α1-containing nAChRs. Basal fluorescence of each well was subtracted from maximal signals, and used to normalize signals from each well. All curves represent averages (± s.d.) of three independent experiments, and each experiment was conducted in triplicates. (F) Cells from the same transient transfections as used for the Ca²⁺ flux assay were immuno-stained and confocal pictures taken at 40x magnifications with inlets depicting one enlarged single cell.