Mechanistic Insights into the Allosteric Modulation of Opioid Receptors by Sodium Ions

The idea of sodium ions altering G-protein-coupled receptor (GPCR) ligand binding and signaling was first suggested for opioid receptors (ORs) in the 1970s and subsequently extended to other GPCRs. Recently published ultra-high-resolution crystal structures of GPCRs, including that of the δ-OR subtype, have started to shed light on the mechanism underlying sodium control in GPCR signaling by revealing details of the sodium binding site. Whether sodium accesses different receptor subtypes from the extra- or intracellular sides, following similar or different pathways, is still an open question. Earlier experiments in brain homogenates suggested a differential sodium regulation of ligand binding to the three major OR subtypes, in spite of their high degree of sequence similarity. Intrigued by this possibility, we explored the dynamic nature of sodium binding to δ-OR, μ-OR, and κ-OR by means of microsecond-scale, all-atom molecular dynamics (MD) simulations. Rapid sodium permeation was observed exclusively from the extracellular milieu, and following similar binding pathways in all three ligand-free OR systems, notwithstanding extra densities of sodium observed near nonconserved residues of κ-OR and δ-OR, but not in μ-OR. We speculate that these differences may be responsible for the differential increase in antagonist binding affinity of μ-OR by sodium resulting from specific ligand binding experiments in transfected cells. On the other hand, sodium reduced the level of binding of subtype-specific agonists to all OR subtypes. Additional biased and unbiased MD simulations were conducted using the δ-OR ultra-high-resolution crystal structure as a model system to provide a mechanistic explanation for this experimental observation

The correlation between actual grain yields and yields predicted using makers linked to QTL for PH on 1H (QPh.NaTx-1H), 2H (QPh.NaTx-2H), 3H(QPh3H) and 7H (QPh7H) (Table S1).

<p>The correlation between actual grain yields and yields predicted using makers linked to QTL for PH on 1H (QPh.NaTx-1H), 2H (QPh.NaTx-2H), 3H(QPh3H) and 7H (QPh7H) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090144#pone.0090144.s003" target="_blank">Table S1</a>).</p

QTL for KW and GY detected in a DH population derived from a TX9425/Naso Nijo cross grown in different environments.

<p>The position is that of the nearest marker; R² means percentage genetic variance explained by the nearest marker.</p><p>*X: No significant QTL was detected.</p

A New QTL for Plant Height in Barley (<i>Hordeum vulgare</i> L.) Showing No Negative Effects on Grain Yield

<div><p>Introduction</p><p>Reducing plant height has played an important role in improving crop yields. The success of a breeding program relies on the source of dwarfing genes. For a dwarfing or semi-dwarfing gene to be successfully used in a breeding program, the gene should have minimal negative effects on yield and perform consistently in different environments.</p><p>Methods</p><p>In this study, 182 doubled haploid lines, generated from a cross between TX9425 and Naso Nijo, were grown in six different environments to identify quantitative trait loci (QTL) controlling plant height and investigate QTL × environments interaction.</p><p>Results</p><p>A QTL for plant was identified on 7H. This QTL showed no significant effects on other agronomic traits and yield components and consistently expressed in the six environments. A sufficient allelic effect makes it possible for this QTL to be successfully used in breeding programs.</p></div

The major QTL on 7H for plant height.

<p>Left: rMQM mapping results; right: MQM mapping results. Only a few selected markers were presented. For detailed map, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090144#pone.0090144.s001" target="_blank">Figure S1</a>. Markers in bold are the nearest markers to the QTL.</p

Characterisation of aroma profiles of commercial sufus by odour activity value, gas chromatography-olfactometry, aroma recombination and omission studies

<p>Sufu is a solid-state fermented product made from soya beans. For the sake of quality control and regulation purposes, it is essential to be able to identify key odorants of various commercial sufus. To identify the aroma-active compounds in sufus, gas chromatography-olfactometry/aroma extract dilution analysis (GC-O/AEDA) was performed, and odour activity value (OAV) was estimated. The correlations between aroma profiles and identified aroma-active compounds were also investigated by principal component analysis. Results showed that 35 aroma-active compounds were detected through OAV calculation, while 28 compounds were identified by using GC-O/AEDA. Quantitative descriptive analysis revealed that aroma recombination model based on OAV calculation was more similar to original sufu in terms of aroma comparing to aroma recombination model based on GC-O/AEDA. Omission experiments further confirmed that the aroma compounds, such as ethyl butanoate, ethyl 2-methylbutanoate, ethyl hexanoate, (<i>E</i>,<i>E</i>)-2,4-decadienal and 2,6-dimethylpyrazine, contributed most significantly to the characteristic aroma of a commercial sufu.</p

QTL for KW and GY detected in a DH population derived from a TX9425/Naso Nijo cross grown in different environments.

<p>The position is that of the nearest marker; R² means percentage genetic variance explained by the nearest marker.</p><p>*X: No significant QTL was detected.</p

QTL for plant height (PH) and heading date (HD).

<p>The number of ‘*’ indicating the number of environments that QTL was detected and ‘-A’ indicating that the QTL was detected based on the average values of all environments. For detail map with markers, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090144#pone.0090144.s001" target="_blank">Figure S1</a>.</p

Early notes

The reactions of the 16-electron half-sandwich complex CpCo­(S₂C₂B₁₀H₁₀) (<b>1</b>) (Cp: cyclopentadienyl) with sulfonyl azides (<i>p</i>-toluenesulfonyl azide, TsN₃; methanesulfonyl azide, MsN₃) in refluxing dichloromethane or at ambient temperature lead to imido-bridged adducts CpCo­(S₂C₂B₁₀H₁₀) (NSO₂R) (<b>2a</b>, R = 4-MePh; <b>2b</b>, R = Me) which can convert to the tetraazadiene cobalt complexes CpCoN₄(SO₂R)₂ (<b>3a</b>, R = 4-MePh; <b>3b</b>, R = Me) in the presence of excess azide if heated. The reactions of <b>1</b> with acyl azides (methyl azidoformate and benzoyl azide) lead to CpCo­(S₂C₂B₁₀H₁₀)­(CONR) (<b>4a</b>, R = OMe; <b>4b</b>, R = Ph) with a newly-generated five-membered metallacyclic ring Co–S–N–C–O. Complexes <b>2a</b> and <b>2b</b> show further reactivity toward alkynes to give rise to the insertion products CpCo­(S₂C₂B₁₀H₁₀)­(R₁CCR₂) (NSO₂R) (R₁ = COOMe, R₂ = H, R = 4-MePh, <b>5a</b>, R = Me, <b>5b</b>; R₁ = R₂ = COOMe, R = 4-MePh, <b>6a</b>, R = Me, <b>6b</b>; R₁ = COOMe, R₂ = Ph, R = 4-MePh, <b>8a</b>, R = Me, <b>8b</b>) formed by alkyne addition to a Co–S bond to generate a Co–C–C–S four-membered ring and CpCo­(S₂C₂B₁₀H₁₀)­(R₁CCR₂NSO₂R) (R₁ = H, R₂ = Ph, R = 4-MePh, <b>7a</b>, R = Me, <b>7b</b>; R₁ = COOMe, R₂ = Ph, R = 4-MePh, <b>9a</b>, R = Me, <b>9b</b>) formed by alkyne insertion into a Co–N bond to generate a Co–C–C–N–S five-membered ring. In the case of PhCCCO₂Me, the products with insertion into both Co–S and Co–N bonds are isolated. Interestingly, if <i>tert</i>-butylacetylene is used, CpCo­(S₂C₂B₁₀H₁₀)­(R₁R₂CCNSO₂R) (R₁ = <i>t</i>Bu, R₂ = H, R = 4-MePh, <b>10a</b>, R = Me, <b>10b</b>) are generated by insertion of terminal carbon into a Co–N bond to form four-membered ring Co–C–N–S. The insertion pathways of these reactions have been discussed on the basis of DFT calculations. All the new complexes were fully characterized, and X-ray structural analyses were performed for <b>2a</b>, <b>3a</b>, <b>3b</b>, <b>4a</b>, <b>4b</b>, <b>5a</b>, <b>6a</b>, <b>7a</b>, <b>7b, 8a</b>, <b>9a</b>, <b>9b</b>, and <b>10b</b>

Proposed Mode of Binding and Action of Positive Allosteric Modulators at Opioid Receptors

Available crystal structures of opioid receptors provide a high-resolution picture of ligand binding at the primary (“orthosteric”) site, that is, the site targeted by endogenous ligands. Recently, positive allosteric modulators of opioid receptors have also been discovered, but their modes of binding and action remain unknown. Here, we use a metadynamics-based strategy to efficiently sample the binding process of a recently discovered positive allosteric modulator of the δ-opioid receptor, BMS-986187, in the presence of the orthosteric agonist SNC-80, and with the receptor embedded in an explicit lipid–water environment. The dynamics of BMS-986187 were enhanced by biasing the potential acting on the ligand–receptor distance and ligand–receptor interaction contacts. Representative lowest-energy structures from the reconstructed free-energy landscape revealed two alternative ligand binding poses at an allosteric site delineated by transmembrane (TM) helices TM1, TM2, and TM7, with some participation of TM6. Mutations of amino acid residues at these proposed allosteric sites were found to either affect the binding of BMS-986187 or its ability to modulate the affinity and/or efficacy of SNC-80. Taken together, these combined experimental and computational studies provide the first atomic-level insight into the modulation of opioid receptor binding and signaling by allosteric modulators