Poly(Ethylene Glycol) as a Scaffold for High-Affinity Open-Channel Blockers of the Mouse Nicotinic Acetylcholine Receptor

<div><p>High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (<b>PQ1</b>–<b>5</b>), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). <b>PQ1</b>–<b>5</b> are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore.</p></div

A comparison of non-PEG-based and PEG-based blockers with respect to affinity and molecular complexity.

<p>Affinity is expressed as log (K_d). Non-PEG-based blockers are shown as red circles, while PEG-based blockers are shown as blue diamonds. The plotted data are obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112088#pone.0112088.s005" target="_blank">Table S1</a> and references therein.</p

Kinetic characterizations of AChR blockade by PQ1–5.

<p>(A) Single-channel currents of the AChR in the presence of 100 µM Ach ±5 µM <b>PQ5</b>. Recordings were carried out in the cell-attached configuration held at +70 mV. Currents are displayed as upward deflections. (B) Dose-dependent decrease in the apparent mean open time (τ_app) of the AChR. Data are plotted as mean ± SEM (n = 3–4). (C) Closed-dwell histograms (duration in ms) at 100 µM ACh in the presence (blue) and absence (red) of 5 µM <b>PQ5</b>. (D) Blocking (filled symbols) and unblocking (open symbols) rates of <b>PQ5</b> estimated by MIL analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112088#pone.0112088-Bouzat1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112088#pone.0112088-Qin1" target="_blank">[11]</a>. Data are plotted as mean ± SEM (n = 3). The blocking rate constant (<i>k_+B</i>) of <b>PQ5</b> can be estimated from the slope of the least-squares linear fit for the blocking rates (black line). (E) The unblocking rate constants (<i>k_–B</i>; mean + SEM, n = 6–8) of <b>PQ1</b>–<b>5</b> for fast (blue) and slow (red) dissociation modes estimated from MIL analysis. (F) The blocking rate constants of <b>PQ1</b>–<b>5</b> for fast (blue) and slow (red) dissociation modes estimated from MIL analysis (as illustrated in panel D). Error bars represent the standard errors of the linear fits. The results of MIL analysis (τ_apps, blocking rates, and unblocking rates at different doses) for <b>PQ1</b>–<b>4</b> are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112088#pone.0112088.s003" target="_blank">Figure S3</a>.</p

Structure and mode of action of PEG-based open-channel blockers (PQ1–PQ5) for the AChR.

<p>Structure and mode of action of PEG-based open-channel blockers (PQ1–PQ5) for the AChR.</p

Design of a Highly Bistable Photoswitchable Tethered Ligand for Rapid and Sustained Manipulation of Neurotransmission

Photoswitchable neurotransmitter receptors are powerful tools for precise manipulation of neural signaling. However, their applications for slow or long-lasting biological events are constrained by fast thermal relaxation of <i>cis</i>-azobenzene. We address this issue by modifying the <i>ortho</i> positions of azobenzene used in the tethered ligand. In cultured cells and intact brain tissue, conjugating inhibitory neurotransmitter receptors with one of the derivatives, <b>dMPC1</b>, allows bidirectional receptor control with 380 and 500 nm light. Moreover, the receptors can be locked in either an active or an inactive state in darkness after a brief pulse of light. This strategy thus enables both rapid and sustained manipulation of neurotransmission, allowing optogenetic interrogation of neural functions over a broad range of time scales

Engineering a Light-Regulated GABA_A Receptor for Optical Control of Neural Inhibition

Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABA_A receptors (GABA_ARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABA_A receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABA_AR-mediated neural inhibition

Obesity disproportionately impacts lung volumes, airflow and exhaled nitric oxide in children

<div><p>Background</p><p>The current literature focusing on the effect of obesity and overweight on lung function and fraction of exhaled nitric oxide (FeNO) in children, particularly among healthy children of non-European descent, remains controversial. Furthermore, whether the relationship of obesity and overweight with lung function and FeNO in children is modified by atopy is unclear. The objective of this study was to examine the effect of excess weight on lung function parameters and FeNO among Asian children, with a particular focus on exploring the potential effect modification by atopy.</p><p>Methods</p><p>We investigated the effect of excess weight on lung function and FeNO in a population sample of 1,717 children aged 5 to 18 years and explored the potential modifying effect of atopy.</p><p>Results</p><p>There were positive associations of body mass index (BMI) z-score with forced vital capacity (FVC), forced expiratory volume in 1 second (FEV₁), peak expiratory flow (PEF), and forced expiratory flow at 25–75% (FEF_25-75) (all <i>P</i><0.001), after controlling for confounders. The beta coefficient for FEV₁ (0.084) was smaller than that for FVC (0.111). In contrast, a negative association was found between BMI z-score and FEV₁/FVC ratio (<i>P</i><0.001) and FeNO (<i>P</i> = 0.03). A consistent pattern of association for lung function variables was observed when stratifying by atopy. There was a negative association of BMI z-score with FeNO in atopic subjects (<i>P</i> = 0.006), but not in non-atopic subjects (<i>P</i> = 0.46).</p><p>Conclusions</p><p>Excess weight disproportionately impacts lung volumes and airflow in children from the general population, independent of atopic status. Excess weight inversely affects FeNO in atopic but not in non-atopic children.</p></div

Multivariable analysis of associations of BMI z-scores with lung function variables and FeNO^*.

<p>Multivariable analysis of associations of BMI z-scores with lung function variables and FeNO^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174691#t004fn002" target="_blank">*</a>}.</p

Demographic and clinical characteristics of study subjects.

<p>Demographic and clinical characteristics of study subjects.</p

Association of BMI categories with sex, age and asthma, respectively^*.

<p>Association of BMI categories with sex, age and asthma, respectively^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174691#t002fn002" target="_blank">*</a>}.</p