Amplitude and desensitization of nAChR responses examined at different temperatures.

†, ††<p>Data for desensitization of all receptor and agonist combinations (with the exception of wild-type α7, activated by ACh) are expressed as the percentage of decay from the peak response in 5 seconds. Due to the rapid rate of desensitization for wild-type α7 activated by ACh, these values are expressed as the time required for the response to decay to 50% of the peak response.</p><p>Data are means ± SEM. Significant differences to responses recorded at RT are indicated (* = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001).</p

Electrophysiological characterization of α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to acetylcholine.

<p>Bar charts illustrate responses (mean ± SEM) from α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to a maximal (3 mM) and <i>EC</i>₅₀ (100 µM) concentration of acetylcholine (A and B, respectively) at room temperature (RT; 21°C), higher temperature (37°C) and lower temperature (4°C). Data are means of 7–11 responses, each from a different oocyte, in which responses obtained at either 4°C or 37°C are normalized to responses obtained from the same oocyte at RT. C) Representative traces illustrating responses obtained at RT (upper trace) and 4°C (lower trace) from a single oocyte. D) Representative traces illustrating responses obtained at RT (upper trace) and 37°C (lower trace) from a single oocyte.</p

Electrophysiological characterization of α7^L247T nAChRs expressed in <i>Xenopus</i> oocytes in response to acetylcholine.

<p>Bar charts illustrate responses (mean ± SEM) from α7^L247T nAChRs expressed in <i>Xenopus</i> oocytes in response to a maximal (30 µM) and <i>EC</i>₅₀ (0.4 µM) concentration of acetylcholine (A and B, respectively) at room temperature (RT; 21°C), higher temperature (37°C) and lower temperature (4°C). Data are means of 5–9 responses, each from a different oocyte, in which responses obtained at either 4°C or 37°C are normalized to responses obtained from the same oocyte at RT. C) Representative traces illustrating responses obtained at RT (upper trace) and 4°C (lower trace) from a single oocyte. D) Representative traces illustrating responses obtained at RT (upper trace) and 37°C (lower trace) from a single oocyte.</p

Electrophysiological characterization of nAChRs expressed in <i>Xenopus</i> oocytes examined at different temperatures.

<p>Representative traces are shown illustrating responses obtained at RT (black), 4°C (blue) and 37°C (red). Current traces obtained at each temperature have been normalized to the same peak response. In each case, the response showing the fastest rate of desensitization was observed at 37°C and the slowest rate of desensitization was observed at 4°C. Responses are from α7 nAChRs with 3 mM acetylcholine (A), α4β2 nAChRs with 1 mM acetylcholine in calcium-containing saline (B), α7^L247T nAChRs with 30 µM acetylcholine (C) and α7 nAChRs with 10 µM 4BP-TQS (D). Rates of receptor deactivation after removal of agonist were also influenced in a consistent manner by changes in temperature (faster at 37°C and slower at 4°C). Representative traces from α7^L247T nAChRs with 30 µM acetylcholine are illustrated (E) and are typical of results from all receptor/agonist combinations studied (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032073#pone-0032073-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032073#pone-0032073-t002" target="_blank">2</a> for details).</p

Electrophysiological characterization of α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to 4BP-TQS.

<p>A) A bar chart illustrates responses (mean ± SEM) from α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to a maximal (10 µM) concentration of the allosteric agonist 4BP-TQS at room temperature (RT; 21°C), higher temperature (37°C) and lower temperature (4°C). Data are means of 5–22 responses, each from a different oocyte, in which responses obtained at either 4°C or 37°C are normalized to responses obtained from the same oocyte at RT. B) Representative traces illustrating responses obtained at RT (upper trace) and 4°C (lower trace) from a single oocyte. C) Representative traces illustrating responses obtained at RT (upper trace) and 37°C (lower trace) from a single oocyte.</p

Characterisation of TQS and 4BP-TQS in human induced pluripotent stem cell-derived neurons examined by fluorescence-based intracellular calcium imaging.

<p>A) Pre- and co-application of TQS (30 µM) with compound B (30 nM) potentiates the amplitude of compound B -evoked calcium-responses (almost undetectable in the absence of TQS) (Left). The trace represents the average response of 35 individual traces ± SEM. Responses to compound B (30 µM) co-applied with TQS (30 µM) are blocked when pre- and co-applied with MLA (1 µM; Right). The trace represents the average response of 137 individual traces ± SEM. B) Agonist-evoked calcium-responses were detectable in response to the allosteric agonist 4BP-TQS (30 µM; Left). The trace represents the average response of 122 individual traces ± SEM. Responses to 4BP-TQS (30 µM) were blocked by pre- and co-application of MLA (1 µM; Right). The trace represents the average response of 118 individual traces 5± SEM.</p

Positive allosteric modulation of α7 nAChRs by TQS examined by patch-clamp in rat primary hippocampal cells.

<p>A) Representative recordings showing responses to the application of compound B (1 µM; Left; black bar) and of TQS (10 µM; grey bar) pre-applied for 5 s and then co-applied with compound B (1 µM; Right; black bar). B) Co-application of compound B (1 µM; black bar) with TQS (10 µM; grey bar) results in a two-component response.</p

Subtype selectivity of compound B, TQS and 4BP-TQS examined with human recombinant nAChR subtypes expressed in <i>Xenopus</i> oocytes.

<p>A) Responses to ACh (3 mM on α7 nAChRs and 100 µM on other nAChR subtypes; black bars) and to compound B (30 µM; grey bars). B) Responses to ACh (100 µM) and to ACh (100 µM; black bars) pre- and co-applied with TQS (100 µM; grey bars). C) Responses to ACh (3 mM on α7 nAChRs and 100 µM on other nAChR subtypes; black bars) and to 4BP-TQS (60 µM on α7 nAChRs and 100 µM on other nAChR subtypes; grey bars).</p

Characterisation of native nAChRs in rat primary hippocampal cells and human induced pluripotent stem cell-derived neurons, examined by fluorescence-based intracellular calcium imaging.

<p>A) Pseudocolour images of rat hippocampal neurons corresponding to low initial resting calcium levels (Left) and higher calcium levels, after application of 30 µM 4BP-TQS (Right). B) Single cell traces (cyan) for all neurons present in the optical field. The average response is shown in red (n = 89 cells). C) Histogram illustrating the percentage of cells that responded to compound B (1 µM), compound B co-applied with TQS (1 µM and 10 µM, respectively) and 4BP-TQS (30 µM) in rat primary hippocampal cells (blue) and in iCell neurons (red). Data were normalised to the total number of cells that responded to KCl (50 mM) (n = 3–31).</p

Pharmacological properties of TQS and 4BP-TQS on nAChR subtypes.

*<p>Fold potentiation of response to ACh (100 µM) by TQS (100 µM).</p>**<p>Percentage inhibition of response to ACh (100 µM) by TQS (100 µM).</p>†<p>Agonist response of 4BP-TQS (100 µM) expressed as a fold response normalised to maximal concentration of ACh (3 mM).</p>††<p>Percentage inhibition of response to ACh (100 µM) by 4BP-TQS (100 µM). Data are means ± SEM.</p