Kinetic analysis of recombinant mammalian α<Subscript>1</Subscript> and α<Subscript>1</Subscript>β glycine receptor channels

To analyze the influence of the beta-subunit on the kinetic properties of GlyR channel currents, alpha(1)-subunits and alpha(1)beta-subunits were transiently expressed in HEK 293 cells. A piezo dimorph was used for fast application of glycine to outside-out patches. The rise time of activation was dose dependent for both receptors and decreased with increasing glycine concentrations. Subunit composition had no effect on the time course of activation. Coexpression of alpha(1)- and beta-subunits resulted in a significantly lower EC(50) and a reduced slope of the dose-response curve of glycine compared with expression of alpha(1)-subunits alone. For both receptor subtypes, the time course of desensitization was concentration dependent. Desensitization was best fitted with a single time constant at 10-30 micro M, with two at 0.1 mM, and at saturating concentrations (0.3-3 mM) with three time constants. Desensitization of homomeric alpha(1)-receptor channels was significantly slower than that of alpha(1)beta-receptor channels. The time course of current decay after the end of glycine pulses was tested at different pulse durations of 1 mM glycine. It was best fitted with two time constants for both alpha(1) and alpha(1)beta GlyR channels, and increased significantly with increasing pulse duration.     

Presence of Ornithine in the Urate-binding α<Subscript>1</Subscript>—<Subscript>2</Subscript>Globulin

THE urate-binding α₁–α₂ globulin has been isolated from human plasma in a highly purified state¹. The protein was purified by DEAE-‘Sephadex’, ammonium sulphate precipitation and semi-preparative Polyacrylamide gel electrophoresis. The urate-binding α₁–α₂ globulin is a rod-shaped glycoprotein, containing 12.1% carbohydrate, with an isoelectric point of 4.6 and a molecular weight of 67,000 ± 4,000. Amino-acid analysis indicated an unknown basic compound which appeared as an extra peak just in front of lysine¹. To identify this compound, high voltage paper electrophoresis has been carried out on a plate electrophoresis apparatus in pyridine-acetate buffer pH 3.5. A spot separated out corresponding to ornithine. Amino-acid analysis on a BC-200 automatic analyser (Bio-Cal Instruments Co., West Germany), with a 54 cm column at 55° C and with 0.35 M sodium citrate buffer, pH 5.28, as elution buffer at a flow-rate of 150 ml./h, showed that ornithine was present. The presence of ornithine in the protein hydrolysate was also verified by gas chromatography/mass spectrometry².     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, ₂-microglobulin (₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/₂m dimers. Association of mouse MHC class I heavy chain with ₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with ₂m are different for each allele. While searching for the PLC binding site in the ₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, ₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse ₂m. Thus, there are allelic differences in the modes of binding of ₂m to the heavy chain of MHC class I.     

Functional blockade of α<Subscript>5</Subscript>β<Subscript>1</Subscript> integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

Background  

Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α₅β₁ integrin in liver progenitor cells.     

Different effects of SNP and GSNO on mitochondrial O<Stack><Subscript>2</Subscript><Superscript>.−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> production

Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) differently affect mitochondrial H₂O₂ release at Complex-I. mM SNP increases while GSNO decreases the release induced by succinate alone or added on top of NAD-linked substrates. Stimulation likely depends on Nitric Oxide ( ^. NO) (released by SNP but not by GSNO) inhibiting cytochrome oxidase and mitochondrial respiration. Preincubations with SNP or high GSNO (10 mM plus DTE to increases its ^. NO release) induces an inhibition of the succinate dependent H₂O₂ production consistent with a ^. NO dependent covalent modification. However maximal inhibition of the succinate dependent H₂O₂ release is obtained in the presence of low GSNO (20–100 μM), but not with SNP. This inhibition appears independent of ^. NO release since μM GSNO does not affect mitochondrial respiration, or the H₂O₂ detection systems and its effect is very rapid. Inhibition may be partly due to an increased removal of O₂^.− since GSNO chemically competes with NBT and cytochrome C in O₂^.− detection.     

Hydrogen-bonding behavior of various conformations of the HNO<Subscript>3</Subscript>…(CH<Subscript>3</Subscript>OH)<Subscript>2</Subscript> ternary system

Nine minima were found on the intermolecular potential energy surface for the ternary system HNO₃(CH₃OH)₂ at the MP2/aug-cc-pVDZ level of theory. The cooperative effect, which is a measure of the hydrogen-bonding strength, was probed in these nine conformations of HNO₃…(CH₃OH)₂. The results are discussed here in terms of structures, energetics, infrared vibrational frequencies, and topological parameters. The cooperative effect was observed to be an important contributor to the total interaction energies of the cyclic conformers of HNO₃…(CH₃OH)₂, meaning that it cannot be neglected in simulations in which the pair-additive potential is applied.

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Graphical abstract The H-bonding behavior of various conformations of the HNO₃(CH₃OH)₂ trimer was investigated

     

Structures and stability of N<Subscript>13</Subscript><Superscript>+</Superscript> and N<Subscript>13</Subscript><Superscript>−</Superscript> clusters

The structures and stabilities of eleven N₁₃ ⁺ and N₁₃ ^– isomers have been investigated with second-order Møller–Plesset (MP2) and density functional theory (DFT) methods. Five N₁₃ ⁺ isomers and six N₁₃ ^– isomers are all reasonable local minima on their potential energy hypersurfaces. The most stable N₁₃ ⁺ cation is structure C-2 with C_2v symmetry, which contains a pentazole ring and two N₄ open chains. It is different from those of the N₇ ⁺ and N₉ ⁺ clusters, but similar to the N₁₁ ⁺ cluster. Meanwhile, the most stable N₁₃ ^– structure A-2 is composed of a pentazole ring and a six-membered ring connected by two nitrogen atoms. It is not only different from those of the N₇ ^– and N₉ ^– clusters, but also from the N₁₁ ^– cluster. The decomposition pathways of structures C-2 and A-2 were investigated at the B3LYP/(aug)-cc-pVDZ level. From the barrier heights of the structures C-2 and A-2 decomposition processes, it is suggested that C-2 is difficult to observe experimentally and A-2 may be observed as a short-lived species. Figure Optimized geometrical parameters of N₁₃ ⁺ isomer C-2      

The neural γ<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript> gamma amino butyric acid ion channel receptor: structural analysis of the effects of the ivermectin molecule and disulfide bridges

While ~30% of the human genome encodes membrane proteins, only a handful of structures of membrane proteins have been resolved to high resolution. Here, we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors, human type A γ₂α₁β₂α₁β₂ gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy, ataxia, migraine, schizophrenia and other neurodegenerative diseases. The structure of human γ₂α₁β₂α₁β₂ has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here, our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100 ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule, ivermectin, from the template structure, helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts, there are three potential disulfide bridges between the M1 and M3 helices of the γ₂ and 2 α₁ subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models—one with the disulfide bridge and one with protonated Cys residues. In all subunit types, RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally, loop A, loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory, the two model structures displayed different coupling in between the M2–M3 linker region, protruding from the membrane, and the β1-β2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop, which collapses directly over the bound ligand molecule, were also affected by differences in the packing of transmembrane helices. Finally, more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.     

Rho GTPases mediated integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> activation in sphingosine-1-phosphate stimulated chemotaxis of endothelial cells

Integrins, a family of transmembrane heterodimeric polypeptides, mediate various biological responses including cell adhesion and migration. In this report, we show that sphingosine-1-phosphate (S1P) activates integrin α_vβ₃ in endothelial cells (ECs) via the sphingosine-1-phosphate receptor subtype 1 (S1P1)-mediated signaling pathway. S1P treatment results in the activation of integrin α_vβ₃ in the lamellipodia region of ECs, suggesting that integrin α_vβ₃ plays a critical role in the S1P-stimulated chemotactic response of ECs. Indeed, S1P treatment induces the association of focal adhesion kinase (FAK) and cytoskeletal proteins with integrin α_vβ₃, the ligation of α_v and β₃ subunits, as well as enhances endothelial migration on vitronectin-coated substrata. Knockdown endothelial S1P1 receptor, treatments with pertussis toxin or dominant-negative-Rho family GTPases abrogates the S1P-induced integrin α_vβ₃ activation in ECs. Consequently, these treatments markedly inhibit the S1P-induced endothelial migratory response on vitronectin-coated substrata. Collectively, these data indicate that the S1P-mediated signaling via the S1P1/G_i/Rho GTPases pathway activates integrin α_vβ₃, which is indispensable for S1P-stimulated chemotactic response of ECs.     

PI3-kinase is essential for ADP-stimulated integrin α<Subscript>IIb</Subscript>β<Subscript>3</Subscript>-mediated platelet calcium oscillation,implications for P2Y receptor pathways in integrin α<Subscript>IIb</Subscript>β<Subscript>3</Subscript>-initiated signaling cross-talks

Summary Phosphatidylinositol 3-kinase (PI3K) pathway is important for platelet activation. Recent studies showed that PI3K and oscillative calcium could cross talk to each other and positively regulate integrin α _IIbβ₃-mediated outside-in signaling. However, the mechanism of this feedback regulation remains to be further characterized. Here we found that treatments of both PI3K inhibitor wortmannin and P2Y1 inhibitor A3P5P could inhibit granular secretion in platelets. Additionally, when RGD-substrate adherent platelets were treated with the ADP scavenger apyrase to deplete the granular-released ADP, their attachments in engaging with substrates became looser and the frequency of calcium oscillation decreased. Since it is known that ADP stimulates the PI3K and calcium signal primarily through P2Y12 and P2Y1 receptors respectively, our data indicated that integrin α_IIbβ₃ downstream PI3K and calcium activation might be not completely coupled to integrin associated signaling complex, but in part through feedback stimulation by granular released ADP. Our data indicates the important roles of PI3K and granular released ADP in coordinating the feedback regulations in integrin α_IIbβ₃-mediated platelet activation.     

Thyroid hormone receptor α<Subscript>1</Subscript>–β<Subscript>1</Subscript> expression in epididymal epithelium from euthyroid and hypothyroid rats

The objectives of the present work were to assess whether epithelial cells from the different segments of epididymis express TRα₁–β₁ isoforms, to depict its subcellular immunolocalization and to evaluate changes in their expression in rats experimentally submitted to a hypothyroid state by injection of 131I. In euthyroid and hypothyroid groups, TR protein was expressed in epididymal epithelial cells, mainly in the cytoplasmic compartment while only a few one showed a staining in the nucleus as well. A similar TR immunostaining pattern was detected in the different segments of the epididymis. In hypothyroid rats, the number of TR-immunoreactive epithelial cells as well as the intensity of the cytoplasmic staining significantly increased in all sections analyzed. In consonance to the immunocytochemical analysis, the expression of TRα₁–β₁ isoforms, assessed by Western blot revealed significantly higher levels of TR in cytosol compared to the nuclear fractions. Furthermore, TR expression of both α₁ and β₁ isoforms and their mRNA levels were increased by the hypothyroid state. The immuno-electron-microscopy showed specific reaction for TR in principal cells associated with eucromatin, cytosolic matrix and mitochondria. The differences in expression levels assessed in control and thyroidectomized rats ascertain a specific function of TH on this organ.     

Computational studies of the binding site of α<Subscript>1A</Subscript>-adrenoceptor antagonists

Aimed at achieving a good understanding of the 3-dimensional structures of human α_1A-adrenoceptor (α_1A-AR), we have successfully developed its homology model based on the crystal structure of β₂-AR. Subsequent structural refinements were performed to mimic the receptor’s natural membrane environment by using molecular mechanics (MM) and molecular dynamics (MD) simulations in the GBSW implicit membrane model. Through molecular docking and further simulations, possible binding modes of subtype-selective α_1A-AR antagonists, Silodosin, RWJ-69736 and (+)SNAP-7915, were examined. Results of the modeling and docking studies are qualitatively consistent with available experimental data from mutagenesis studies. The homology model built should be very useful for designing more potent subtype-selective α_1A-AR antagonists and for guiding further mutagenesis studies. Figure The superposition of β₂-AR crystal structure (gold ribbons) and α_1A-AR homology model (blue ribbons)     

Insight into π-hole interactions containing the inorganic heterocyclic compounds S<Subscript>2</Subscript>N<Subscript>2</Subscript>/SN<Subscript>2</Subscript>P<Subscript>2</Subscript>

Similar to σ-hole interactions, the π-hole interaction has attracted much attention in recent years. According to the positive electrostatic potentials above and below the surface of inorganic heterocyclic compounds S₂N₂ and three SN₂P₂ isomers (heterocyclic compounds 1–4), and the negative electrostatic potential outside the X atom of XH₃ (X = N, P, As), S₂N₂/SN₂P₂?XH₃ (X = N, P, As) complexes were constructed and optimized at the MP2/aug-cc-pVTZ level. The X atom of XH₃ (X = N, P, As) is almost perpendicular to the ring of the heterocyclic compounds. The π-hole interaction energy becomes greater as the trend goes from 1?XH₃ to 4?XH₃. These π-hole interactions are weak and belong to “closed-shell” noncovalent interactions. According to the energy decomposition analysis, of the three attractive terms, the dispersion energy contributes more than the electrostatic energy. The polarization effect also plays an important role in the formation of π-hole complexes, with the contrasting phenomena of decreasing electronic density in the π-hole region and increasing electric density outside the X atom of XH₃ (X = N, P, As).

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Graphical abstract Computed density difference plots for the complexes 3?NH ₃ (a ₁), 3?PH ₃ (b ₁), 3?AsH ₃ (c ₁) and electron density shifts for the complexes 3?NH ₃ (a ₂), 3?PH ₃ (b ₂),3?AsH ₃ (c ₂) on the 0.001 a.u. contour

     

Effects of Prostaglandins F<Subscript>2α</Subscript>, E<Subscript>2</Subscript> and E<Subscript>1</Subscript> on Fertility in Mice

PROSTAGLANDIN (PG) F_2αhas antifertility effects in many species^1–3 but there are conflicting suggestions as to its mechanism of action. For example, it may cause the degeneration of the corpus luteum by decreasing blood flow in the uteroovarian vein⁴; alternatively, its action may be due to a hypersecretion of luteinizing hormone (LH) by the pituitary^3,5. I have investigated the effects of PGF_2α, E₂ and E₁ on pregnancy in mice and examined the mechanism of action of PGF_2α.     

Effects of activation of μ-, κ<Subscript>1</Subscript>-, δ<Subscript>1</Subscript>-, δ<Subscript>2</Subscript>-, and ORL<Subscript>1</Subscript>-receptors on heart resistance to the pathogenic action of delayed ischemia and reperfusion

Different subtypes of opioid receptors (OR) were activated in rats in vivo to study the activation effect on the heart’s resistance to ischemia and reperfusion. It has been established that administration of deltorphin II, a selective δ₂-OR agonist, lowered the infarct size/area at risk index (IS/AAR) by 23%. Naltrexone, naloxone methiodide (an OR inhibitor not penetrating the blood-brain barrier (BBB)), and naltriben (δ₂-antagonist) eliminated the cardioprotective effect of deltorphin II, while BNTX (a δ₁-antagonist) produced no effect on the cardioprotective action of the δ₂-agonist. The infarct-reducing effect of deltorphin II was eliminated by administration of chelerythrine (a protein kinase C (PKC) inhibitor), glibenclamide (a K_ATP-channels inhibitor), and 5-hydroxydecanoate (a mitochondrial K_ATP-channel blocker). Administration of other opioids did not reduce the IS/AAR index. It has been established that all the deltorphins manifest antiarrhythmic potency. Other opioids do not produce any effect on the incidence of arrhythmia occurrences. The antiarrhythmic effect of deltorphin II was eliminated by preliminary administration of naltrexone, naloxone methiodide, and naltriben, but BNTX did not affect the δ₂-agonist’s anti-arrhythmic effect. The preliminary administration of chelerythrine, a PKC inhibitor, eliminated the δ₂ agonist’s antiarrhythmic action. However, glibenclamide and 5-hydroxydecanoate did not alter the antiarrhythmic effect by deltorphin II. Therefore, activation of the peripheral δ₂-ORs reduces the infarct size and prevents the onset of arrhythmias. The antiarrhythmic effect of the δ₂-OR stimulation is mediated by activating PKC and opening the mitochondrial K_ATP-channels. PKC participates in the antiarrhythmic effect of the δ₂-OR activation, but this effect does not depend on the condition of K_ATP-channels.     

Deciphering the binding mode of Zolpidem to GABA<Subscript>A</Subscript> α<Subscript>1</Subscript> receptor – insights from molecular dynamics simulation

To investigate the binding mode of Zolpidem to GABA(A) and to delineate the conformational changes induced upon agonist binding, we carried out atomistic molecular dynamics simulation using the ligand binding domain of GABA(A) α(1) receptor. Comparative molecular dynamics simulation of the apo and the holo form of GABA(A) receptor revealed that γ(2)/α(1) interface housing the benzodiazepine binding site undergoes distinct conformational changes upon Zolpidem binding. We notice that C loop of the α(1) subunit experiences an inward motion toward the vestibule and the F loop of γ(2) sways away from the vestibule, an observation that rationalizes Zolpidem as an alpha1 selective agonist. Energy decomposition analysis carried out was able to highlight the important residues implicated in Zolpidem binding, which were largely in congruence with the experimental data. The simulation study disclosed herein provides a meaningful insight into Zolpidem-GABA(A)R interactions and helps to arrive at a binding mode hypothesis with implications for drug design.     

Negative molecular ion of deuterium D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack>

D₂⁻ ions produced in collisions of D⁻ ions with relative energies of 2.5–9.2 eV were detected for the first time. It is shown that the effective cross section for this reaction is no less than 1.5 × 10⁻¹⁴ cm². Along with the theoretically predicted short-lived state of negative molecular deuterium ions, a state existing for more than 1 μs was observed.     

Succinate is the controller of O<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at mitochondrial complex I : negative modulation by malate,positive by cyanide

Mitochondrial production of H₂O₂ is low with NAD substrates (glutamate/pyruvate, 3 and 2 mM) (G/P) and increases over ten times upon further addition of succinate, with the formation of a sigmoidal curve (semimaximal value at 290 μM, maximal H₂O₂ production at 600 μM succinate). Malate counteracts rapidly the succinate induced increased H₂O₂ release and moves the succinate dependent H₂O₂ production curve to the right. Nitric oxide (NO) and carbon monoxide (CO) are cytochrome c oxidase inhibitors which increase mitochondrial ROS production. Cyanide (CN⁻) was used to mimic NO and CO. In the presence of G/P and succinate (300 μM), CN⁻ progressively increased the H₂O₂ release rate, starting at 1.5 μM. The succinate dependent H₂O₂ production curve was moved to the left by 30 μM CN⁻. The V_max was little modified. We conclude that succinate is the controller of mitochondrial H₂O₂ production, modulated by malate and CN⁻. We propose that succinate promotes an interaction between Complex II and Complex I, which activates O₂⁻ production.