Effects of the 9′ LT mutation on the ACh dose-response curve of human α3β4 receptors expressed in oocytes at extreme ratios.

<p><i>EC</i>₅₀, <i>I_max</i> and Hill slope are means ± standard error of the mean of separate fits of each dose-response curve to the Hill equation. Potency ratios are expressed relative to the wild-type curve and were estimated from parallel fits to the Hill equation (note that the dose-response curve for the 1∶9 α3β4^LT receptor was too shallow to allow parallel fits do be done without too much distortion, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013611#pone.0013611-Boorman1" target="_blank">[13]</a>. The 2-unit likelihood intervals for the potency ratios are shown in brackets.</p

Single-channel properties of α3β4 receptors expressed from extreme ratios in HEK293 cells.

<p>(A) Examples of outside-out currents elicited by 5 µM ACh, from cells transfected with an α3 to β4 cDNA ratio of 1∶9 (left) and 9∶1 (right). Patches were held at −100 mV. The histograms of fitted amplitudes corresponding to these recordings are shown in (B). These are fitted with Gaussian curves to give the peak current amplitudes and the areas under each curve (for these two patches the values are 2.7±0.3 pA, area 100% for 1∶9; 2.6±0.5 pA, area 27% and 3.9±0.4 pA, area 73% for 9∶1). The histogram on the left (C) shows the proportion of bursts (as % of the total number of bursts from all experiments, pooled) at each chord conductance for the two subunit ratios. The histogram on the right (D) shows the difference in the duration of the bursts to different conductances for the two ratios.</p

The effect of the 9′ LT mutation in oocytes depends on the α3∶β4 subunit ratio.

<p>Traces in (A) represent inward currents elicited by different concentrations of ACh (in µM) bath applied (duration shown as a solid bar). Cells were held at −70 mV. Pooled normalised dose-response curves, fitted with the Hill equation with free parameters are shown in (B) for oocytes injected with 1∶9 (left) or 9∶1 (right) α3 to β4 cRNA ratio. The 1∶9 subunit ratio produces wild-type receptors that are more sensitive to ACh. The effect of the mutation is greater when it is carried by the β4 subunit if β4 is overexpressed (1∶9) and when the α3 subunit is mutated if the α3 subunit is overexpressed (9∶1).</p

The two-α and three-α form of the α3β4 receptor differ in their response to Zn²⁺ modulation.

<p>Traces in (A) are typical currents activated by an <i>EC</i>₂₀ concentration of ACh on the two-α receptor (left, 1∶9 α∶β ratio) and the three-α receptor (right, 9∶1 ratio) in the presence of increasing concentrations of Zn²⁺. Zn²⁺ was pre-applied to oocytes for 30 s before it was applied together with ACh. (B) Zn²⁺ concentration-response curves for the three-α receptor (filled squares) and the two-α receptor (filled circles). Curves from different cells were pooled and fitted to the Hill equation and to the sum of two Hill equations, respectively. Note that Zn²⁺ potentiated only the ACh responses of oocytes expressing the two-α stoichiometry.</p

Potency ratio values for a range of nicotinic agonists on human α3β4 neuronal nicotinic receptors expressed in oocytes.

<p>Numbers shown are average values ± standard error of the mean. Potency ratios, relative to the standard agonist ACh, express how much more potent than ACh an agonist is at a comparable level of response. The 2-unit likelihood intervals for the potency ratios are shown in brackets.</p

In HEK-expressed α3β4 receptors, the 9′LT mutation has a greater effect if inserted in α3.

<p>Traces in (A) are whole cell inward currents elicited by different ACh concentrations applied to cells expressing wild type (top), α3β4^LT (middle) or α3^LTβ4 (bottom) channels. The bars above the traces show the duration of the agonist applications and the agonist concentration (in µM). Cells were transfected with an α3 to β4 ratio of 1∶1 and held at −30 mV. Dose-response curves are shown in (B) for each receptor combination. The data were averaged after normalisation to the fitted maximum for each experiment. The lines show Hill equation fits (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013611#s4" target="_blank">Methods</a>) to the pooled normalised data (<i>n</i> = 6–8).</p

The two different receptor stoichiometries expressed in oocytes differ in their DMPP sensitivity.

<p>The traces in (A) are examples of inward currents elicited by low agonist concentrations (in µM) and recorded from oocytes injected with a subunit ratio of 1∶9 (top traces) and 9∶1 (bottom). The duration of each agonist application is shown above each trace (solid bar). All agonists (CCh, carbachol; EPB epibatidine; Nic, nicotine; DMPP, dimethylphenylpiperazinium; Cyt, cytisine; LOB, lobeline) were tested on the same oocyte (held at −70 mV), with the exception of lobeline (shown on the right, recorded from a different cell but with similar initial ACh current as the cell on the left, for both subunit ratios). The log-log plots in (B) are partial dose-response curves for the experiments shown in A (left). Note the increase in the potency of DMPP (10-fold leftward shift of the dose-response curve) in the 9∶1 ratio.</p

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

<div><p>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, <i>e</i>.<i>g</i>. 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.</p></div

SMRT analysis of all α1-V275 mutants in the library.

<p>SMRT analysis of all α1-V275 mutants in the library.</p

Principles of SMRT sequencing and other NGS methods.

<p>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.</p