γ-Aminobutyric acid type A (GABAA) receptor activation modulates tau phosphorylation

Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABA(A) receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABA(A) receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABA(A) receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity.     

Arachidonic acid inhibition of L-type calcium (CaV1.3b) channels varies with accessory CaVβ subunits

Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca²⁺ channels by an unknown mechanism at an unknown site. The Ca²⁺ channel pore-forming subunit (Ca_Vα₁) is a candidate for the site of AA inhibition because T-type Ca²⁺ channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory Ca_Vβ subunits on the inhibition of Ca_V1.3b L-type (L-) current by AA. Whole cell Ba²⁺ currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of −10 mV from a holding potential of −90 mV. A one-minute exposure to 10 µM AA inhibited currents with β_1b, β₃, or β₄ 58, 51, or 44%, respectively, but with β_2a only 31%. At a more depolarized holding potential of −60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes Ca_V1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential–dependent inactivation or on recovery from inactivation regardless of Ca_Vβ subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for Ca_V2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of β_2a interfere with inhibition by AA. Our novel findings that the Ca_Vβ subunit alters kinetic changes and magnitude of inhibition by AA suggest that Ca_Vβ expression may regulate how AA modulates Ca²⁺-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.     

Thyroid hormone receptor α1 is a critical regulator for the expression of ion channels during final differentiation of outer hair cells

Cochlear outer hair cells (OHCs) terminally differentiate prior to the onset of hearing. During this time period, thyroid hormone (TH) dramatically influences inner ear development. It has been shown recently that TH enhances the expression of the motor protein prestin via liganded TH receptor β (TRβ) while in contrast the expression of the potassium channel KCNQ4 is repressed by unliganded TRα1. These different mechanisms of TH regulation by TRα1 or TRβ prompted us to analyse other ion channels that are required for the final differentiation of OHCs. We analysed the onset of expression of the Ca²⁺ channel Ca_V1.3, and the K⁺ channels SK2 and BK and correlated the results with the regulation via TRα1 or TRβ. The data support the hypothesis that proteins expressed in rodents prior to or briefly after birth like Ca_V1.3 and prestin are either independent of TH (e.g. Ca_V1.3) or enhanced through TRβ (e.g. prestin). In contrast, proteins expressed in rodents later than P6 like KCNQ4 (∼P6), SK2 (∼P9) and BK (∼P11) are repressed through TRα1. We hypothesise that the precise regulation of expression of the latter genes requires a critical local TH level to overcome the TRα1 repression. Harald Winter and Claudia Braig contributed equally to this work.     

Orientation of palmitoylated CaVβ2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation

The G_q-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca²⁺ channel (Ca_V2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which Ca_Vβ is coexpressed with Ca_V2.2. When palmitoylated Ca_Vβ2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when Ca_Vβ3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of Ca_Vβ2a may occupy an inhibitory site on Ca_V2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of Ca_Vβ2a relative to Ca_V2.2 may displace the palmitoyl groups and prevent them from antagonizing AA''s actions, thereby allowing inhibition even in the presence of Ca_Vβ2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the α interacting domain (AID) of Ca_V2.2''s I–II linker. Ca_Vβs bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple Ca_Vβ''s movements to Ca_V2.2 gating. Although Bdel1/β2a currents exhibited more variable enhancement by SP, Bdel2/β2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from Ca_V2.2 coexpressed with Ca_Vβ3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/β2a currents, suggesting that when palmitoylated Ca_Vβ2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that Ca_Vβ2a''s palmitoyl groups directly interact with an inhibitory site on Ca_V2.2 to block N-current inhibition by SP.     

Polyampholyte Nanoparticles Prepared by Self-Complexation of Cationized Poly(γ-glutamic acid) for Protein Carriers

A novel amphoteric poly(amino acid) is synthesized by grafting a cationic amino acid (L-Arg) to γ-PGA to prepare charged NPs. γ-PGA-Arg NPs can be prepared by the self-complexation of a single polymer by intra-/inter-molecular electrostatic interactions when the polymer is dispersed in water. The size and surface charge of the NPs can be regulated by the grafting degree of Arg (41, 56, and 83%). The smallest NPs are obtained at 56% grafting degree of the γ-PGA-Arg copolymer. The 56 and 83% grafting degree NPs are stable for at least 1 week. Depending on their surface charge, these NPs can selectively adsorb anionically or cationically charged proteins.     

Stable reporter cell lines for peroxisome proliferator-activated receptor γ (PPARγ)-mediated modulation of gene expression

Phosphatidylinositol (PtdIns) is phosphorylated at D-3, D-4, and/or D-5 of the inositol ring to produce seven distinct lipid second messengers known as phosphoinositides (PIs). The PI level is temporally and spatially controlled at the cytosolic face of the cellular membrane. Effectors containing PI-binding domains (e.g., PH, PX, FYVE, ENTH, FERM) associate with specific PIs. This process is crucial for the localization of a variety of cell-signaling proteins, thereby regulating intracellular membrane trafficking, cell growth and survival, cytoskeletal organization, and so on. However, quantitative assessments of protein–PI interactions are generally difficult due to insolubility of PIs in aqueous solution. Here we incorporated PIs into a lipid–protein nanoscale bilayer (nanodisc), which is applied for studying the protein–PI interactions using pull-down binding assay, fluorescence polarization, and nuclear magnetic resonance studies, each facilitating fast, quantitative, and residue-specific evaluation of the protein–PI interactions. Therefore, the PI-incorporated nanodisc could be used as a versatile tool for studying the protein–lipid interactions by various biochemical and biophysical techniques.     

Stable reporter cell lines for peroxisome proliferator-activated receptor γ (PPARγ)-mediated modulation of gene expression

Activation of peroxisome proliferator-activated receptor γ (PPARγ) by ligands is associated with beneficial health effects, including anti-inflammatory and insulin-sensitizing effects. The aim of the current study was to develop luciferase reporter gene assays to enable fast and low-cost measurement of PPARγ agonist and antagonist activity. Two reporter gene assays, PPARγ1 CALUX and PPARγ2 CALUX, were developed by stable transfection of U2OS cells with an expression vector for PPARγ1 or PPARγ2 and a pGL3–3xPPRE–tata-luc or pGL4–3xPPRE–tata-luc reporter construct, respectively. PPARγ1 CALUX and PPARγ2 CALUX cells showed similar concentration-dependent luciferase induction upon exposure to the PPARγ agonists rosiglitazone, troglitazone, pioglitazone, ciglitazone, netoglitazone, and 15-deoxy-Δ^12,14-prostaglandin J₂. The potency to induce luciferase decreased in the following order: rosiglitazone > troglitazone = pioglitazone > netoglitazone > ciglitazone. A concentration-dependent decrease in the response to 50 nM rosiglitazone was observed on the addition of PPARγ antagonist GW9662 or T0070907 in both PPARγ1 CALUX and PPARγ2 CALUX cells. The PPARα agonists WY14643 and fenofibrate failed to induce luciferase activity, confirming the specificity of these cell lines for PPARγ agonists. In conclusion, PPARγ1 CALUX and PPARγ2 CALUX cells provide a reliable and useful tool to screen (bio)chemicals for PPARγ agonist or antagonist activity.     

Induction of endoplasmic reticulum-endosome fusion for antigen cross-presentation induced by poly (γ-glutamic acid) nanoparticles

We previously reported that poly (γ-glutamic acid)-based nanoparticles (γ-PGA NPs) are excellent vaccine carriers for inducing efficient cross-presentation in dendritic cells, thereby producing strong antitumor immunity in vivo. Analyzing the mechanism of cross-presentation induced by γ-PGA NPs will be useful toward designing novel vaccine carriers. In this study, we show an intracellular mechanism of efficient cross-presentation induced by OVA-loaded γ-PGA NPs. Cross-presentation induced by γ-PGA NPs depended on cytoplasmic proteasomes and TAP, similar to the classical MHC class I presentation pathway for endogenous Ags. Intracellular behavior analyzed by confocal laser scanning microscopy revealed that encapsulated OVA and γ-PGA accumulated in both the endoplasmic reticulum (ER) and endosome compartments within 2 h. At the same time, electron microscopy analysis clearly showed that intracellular γ-PGA NPs and encapsulated Au NPs were enveloped in endosome-like vesicles, not in the ER. These findings strongly suggest that γ-PGA NPs enhance ER-endosome fusion for cross-presentation. Moreover, inhibition of ER translocon sec61 significantly decreased the γ-PGA NP/OVA-mediated cross-presentation efficiency, indicating that sec61 is important for transporting Ags from the fused ER-endosome to the cytoplasm. These findings imply that the ER-endosome complex is key for the efficient cross-presentation of Ags encapsulated in γ-PGA NPs.     

Rosuvastatin alleviated the liver ischemia reperfusion injury by activating the expression of peroxisome proliferator-activated receptor gamma (PPARγ)

Journal of Bioenergetics and Biomembranes - Liver ischemia and reperfusion could cause serious damage to liver tissues. Abnormal liver function could induce serious damage and threaten human...     

PKCγ is required for ethanol-induced increases in GABA(A) receptor α4 subunit expression in cultured cerebral cortical neurons

Ethanol exposure produces alterations in GABA(A) receptor function and expression associated with CNS hyperexcitability, but the mechanisms of these effects are unknown. Ethanol is known to increase both GABA(A) receptor α4 subunits and protein kinase C (PKC) isozymes in vivo and in vitro. Here, we investigated ethanol regulation of GABA(A) receptor α4 subunit expression in cultured cortical neurons to delineate the role of PKC. Cultured neurons were prepared from rat pups on postnatal day 0-1 and tested after 18?days. GABA(A) receptor α4 subunit surface expression was assessed using P2 fractionation and surface biotinylation following ethanol exposure for 4?h. Miniature inhibitory post-synaptic currents were measured using whole cell patch clamp recordings. Ethanol increased GABA(A) receptor α4 subunit expression in both the P2 and biotinylated fractions, while reducing the decay time constant in miniature inhibitory post-synaptic currents, with no effect on γ2 or δ subunits. PKC activation mimicked ethanol effects, while the PKC inhibitor calphostin C prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression. PKCγ siRNA knockdown prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression, but inhibition of the PKCβ isoform with PKCβ pseudosubstrate had no effect. We conclude that PKCγ regulates ethanol-induced alterations in α4-containing GABA(A) receptors.     

Development of ligands at γ-aminobutyrric acid type A (GABAA) receptor subtype as new agents for pain relief

The identification of compounds with selective anxiolytic-like effects, exerted through the benzodiazepine site on γ-aminobutyric acid type A (GABA_A) receptors, and that show pronounced antihyperalgesia in several pain models, has oriented research towards the development of new agents for the relief of pain. Starting from our previously reported ligands at the benzodiazepine site on GABA_A receptors showing selective anxiolytic-like effects, we have designed new compounds with the aim of identifying those devoid of the typical side effects of the classical benzodiazepines. Our preliminary results indicate that compounds 4, 10(±) and 11 have a very promising antihyperalgesic profile in different animal pain models (peripheral mono-neuropathy, STZ-induced hyperalgesia). In particular 11 exhibits high potency since it exerted its protective effect starting from the dose of 3 mg/kg po, after single injection.     

α(2)-adrenoceptor agonist-induced inhibition of gastric motor activity is mediated by α(2A)-adrenoceptor subtype in the mouse

The role of α(2)-adrenoceptors in regulation of gastric motility has been well documented. However, only few data are available on the adrenoceptor subtype that mediates this effect. The purpose of the present work was to identify the α(2)-adrenoceptor subtype(s) responsible for the inhibition of gastric motor activity in isolated fundus strip of the mouse. It was shown that (i) the electrically evoked contraction of the gastric fundus strip of the mouse was inhibited by the non-selective α(2)-adrenoceptor stimulant clonidine (EC(50): 0.019±0.001μM), the α(2A)-adrenoceptor subtype selective agonist oxymetazoline (EC(50): 0.004±0.001μM) and the α(2B)-adrenoceptor subtype preferring ST-91 (EC(50): 0.029±0.004μM), (ii) the inhibitory effect of clonidine (1μM), oxymetazoline (0.1μM) and ST-91 (1μM) on the contractions of gastric fundus strip was reversed by the non-selective α(2)-adrenoceptor antagonist idazoxan and α(2A)-adrenoceptor antagonist BRL 44408, but not by the α(2B/2C)-adrenoceptor antagonist ARC-239. (iii) Clonidine and ST-91 inhibited the electrically induced gastric contractions in C57BL/6 wild type mice as well as in α(2B)- and α(2C)-adrenoceptor deficient mice in a concentration-dependent manner; however, neither of them was effective in α(2A)-deficient mice. As a conclusion, it was first demonstrated that the inhibitory effect of α(2)-adrenoceptor agonists on the gastric motor activity of isolated stomach strip of the mouse is mediated purely by α(2A)-adrenoceptors.     

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.     

A two-site immunometric assay for the detection of the expression level of α2(C2)-adrenergic receptor produced in Escherichia coli

Human ₂(C2)-adrenergic receptor was expressed in Escherichia coli as a fusion protein with Bacillus circulans var. alcalophilus cyclomaltodextrin glucanotransferase. For the determination of the expression level (0.6 mg of solubilized fusion protein l^–1 of E. coli culture), a two-site immunometric assay based on two monoclonal antibodies with different epitopes was developed.