Monitoring Voltage-Dependent Charge Displacement of Shaker B-IR K+ Ion Channels Using Radio Frequency Interrogation

Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K+ ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ∼120 µM Cu2+ addition to the external bath. Cu2+ is known to bind to the ShB-IR ion channel and inhibit Shaker K+ conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu2+-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains — capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug–protein interactions

Adaptive evolution of the vertebrate skeletal muscle sodium channel

Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Nav). The skeletal muscle Nav (Nav1.4) channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Nav1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Nav1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Nav1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement

Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis

P53 inactivation is often observed in Burkitt's lymphoma (BL) cells due to mutations in the p53 gene or overexpression of its negative regulator, murine double minute-2 (MDM2). This event is now considered an essential part of the oncogenic process. Epstein–Barr virus (EBV) is strongly associated with BL and is a cofactor in its development. We previously showed that nutlin-3, an antagonist of MDM2, activates the p53 pathway in BL cell lines harboring wild-type p53. However, nutlin-3 strongly induced apoptosis in EBV (−) or latency I EBV (+) cells, whereas latency III EBV (+) cells were much more resistant. We show here that this resistance to apoptosis is also observed in latency III EBV (+) lymphoblastoid cell lines. We also show that, in latency III EBV (+) cells, B-cell lymphona 2 (Bcl-2) is selectively overproduced and interacts with Bcl-2-associated X protein (Bax), preventing its activation. The treatment of these cells with the Bcl-2-homology domain 3 mimetic ABT-737 disrupts Bax/Bcl-2 interaction and allows Bax activation by nutlin-3. Furthermore, treatment with these two compounds strongly induces apoptosis. Thus, a combination of Mdm2 and Bcl-2 inhibitors might be a useful anti-cancer strategy for diseases linked to EBV infection

Reduced conditioned fear response in mice that lack Dlx1 and show subtype-specific loss of interneurons

The inhibitory GABAergic system has been implicated in multiple neuropsychiatric diseases such as schizophrenia and autism. The Dlx homeobox transcription factor family is essential for development and function of GABAergic interneurons. Mice lacking the Dlx1 gene have postnatal subtype-specific loss of interneurons and reduced IPSCs in their cortex and hippocampus. To ascertain consequences of these changes in the GABAergic system, we performed a battery of behavioral assays on the Dlx1 mutant mice, including zero maze, open field, locomotor activity, food intake, rotarod, tail suspension, fear conditioning assays (context and trace), prepulse inhibition, and working memory related tasks (spontaneous alteration task and spatial working memory task). Dlx1 mutant mice displayed elevated activity levels in open field, locomotor activity, and tail suspension tests. These mice also showed deficits in contextual and trace fear conditioning, and possibly in prepulse inhibition. Their learning deficits were not global, as the mutant mice did not differ from the wild-type controls in tests of working memory. Our findings demonstrate a critical role for the Dlx1 gene, and likely the subclasses of interneurons that are affected by the lack of this gene, in behavioral inhibition and associative fear learning. These observations support the involvement of particular components of the GABAergic system in specific behavioral phenotypes related to complex neuropsychiatric diseases

Nutlin-3, the small-molecule inhibitor of MDM2, promotes senescence and radiosensitises laryngeal carcinoma cells harbouring wild-type p53

BACKGROUND: Primary radiotherapy (RT) is a mainstay of treatment for laryngeal squamous cell carcinoma (LSCC). Although the cure rates for early (T1) vocal cord tumours are high, RT proves ineffective in up to a third of T3 carcinomas. Moreover, RT is associated with debilitating early- and late-treatment-related toxicity, thus finding means to de-escalate therapy, while retaining/augmenting therapeutic effectiveness, is highly desirable. p53 is a key mediator of radiation responses; we therefore investigated whether Nutlin-3, a small-molecule inhibitor of MDM2 (mouse double minute 2; an essential negative regulator of p53), might radiosensitise LSCC cells. METHODS: We performed clonogenic assays to measure radiosensitivity in a panel of LSCC cell lines (for which we determined p53 mutational status) in the presence and absence of Nutlin-3. RESULTS: LSCC cells harbouring wild-type p53 were significantly radiosensitised by Nutlin-3 (P<0.0001; log-rank scale), and displayed increased cell cycle arrest and significantly increased senescence (P<0.001) in the absence of increased apoptosis; thus, our data suggest that senescence may mediate this increased radiosensitivity. CONCLUSION: This is the first study showing Nutlin-3 as an effective radiosensitiser in LSCC cells that retain wild-type p53. The clinical application of Nutlin-3 might improve local recurrence rates or allow treatment de-escalation in these patients

GABAergic and Cortical and Subcortical Glutamatergic Axon Terminals Contain CB1 Cannabinoid Receptors in the Ventromedial Nucleus of the Hypothalamus

Background: Type-1 cannabinoid receptors (CB1R) are enriched in the hypothalamus, particularly in the ventromedial hypothalamic nucleus (VMH) that participates in homeostatic and behavioral functions including food intake. Although CB1R activation modulates excitatory and inhibitory synaptic transmission in the brain, CB1R contribution to the molecular architecture of the excitatory and inhibitory synaptic terminals in the VMH is not known. Therefore, the aim of this study was to investigate the precise subcellular distribution of CB1R in the VMH to better understand the modulation exerted by the endocannabinoid system on the complex brain circuitries converging into this nucleus. Methodology/Principal Findings: Light and electron microscopy techniques were used to analyze CB1R distribution in the VMH of CB1R-WT, CB1R-KO and conditional mutant mice bearing a selective deletion of CB1R in cortical glutamatergic (Glu-CB1R-KO) or GABAergic neurons (GABA-CB1R-KO). At light microscopy, CB1R immunolabeling was observed in the VMH of CB1R-WT and Glu-CB1R-KO animals, being remarkably reduced in GABA-CB1R-KO mice. In the electron microscope, CB1R appeared in membranes of both glutamatergic and GABAergic terminals/preterminals. There was no significant difference in the percentage of CB1R immunopositive profiles and CB1R density in terminals making asymmetric or symmetric synapses in CB1R-WT mice. Furthermore, the proportion of CB1R immunopositive terminals/preterminals in CB1R-WT and Glu-CB1R-KO mice was reduced in GABA-CB1R-KO mutants. CB1R density was similar in all animal conditions. Finally, the percentage of CB1R labeled boutons making asymmetric synapses slightly decreased in Glu-CB1R-KO mutants relative to CB1R-WT mice, indicating that CB1R was distributed in cortical and subcortical excitatory synaptic terminals. Conclusions/Significance: Our anatomical results support the idea that the VMH is a relevant hub candidate in the endocannabinoid-mediated modulation of the excitatory and inhibitory neurotransmission of cortical and subcortical pathways regulating essential hypothalamic functions for the individual's survival such as the feeding behavior.L. Reguero is in receipt of a Predoctoral Fellowship from the Basque Country Government (BFI 07.286); I. Buceta is in receipt of a Predoctoral Fellowship from the Basque Country University. Dr. Pedro Grandes' laboratory is supported by The Basque Country Government grant GIC07/70-IT-432-07, by Ministerio de Ciencia e Innovacion (SAF2009-07065) and by Red de Trastornos Adictivos, RETICS, Instituto de Salud Carlos III, MICINN, grant RD07/0001/2001. Dr. Giovanni Marsicano's laboratory is supported by AVENIR/INSERM (with the Fondation Bettencourt-Schueller), by ANR (ANR-06-NEURO-043-01), by European Foundation for the Study of Diabetes (EFSD), by the EU-FP7 (REPROBESITY, contract number HEALTH-F2-2008-223713) and European Commission Coordination Action ENINET (contract number LSHM-CT-2005-19063). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Adenyl cyclases and cAMP in plant signaling - past and present

In lower eukaryotes and animals 3'-5'-cyclic adenosine monophosphate (cAMP) and adenyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, have long been established as key components and second messengers in many signaling pathways. In contrast, in plants, both the presence and biological role of cAMP have been a matter of ongoing debate and some controversy. Here we shall focus firstly on the discovery of cellular cAMP in plants and evidence for a role of this second messenger in plant signal transduction. Secondly, we shall review current evidence of plant ACs, analyse aspects of their domain organisations and the biological roles of candidate molecules. In addition, we shall assess different approaches based on search motifs consisting of functionally assigned amino acids in the catalytic centre of annotated and/or experimentally tested nucleotide cyclases that can contribute to the identification of novel candidate molecules with AC activity such as F-box and TIR proteins

Updates on p53: modulation of p53 degradation as a therapeutic approach

The p53 pathway is aberrant in most human tumours with over 50% expressing mutant p53 proteins. The pathway is critically controlled by protein degradation. Here, we discuss the latest developments in the search for small molecules that can modulate p53 pathway protein stability and restore p53 activity for cancer therapy

MDM2 antagonist Nutlin-3a potentiates antitumour activity of cytotoxic drugs in sarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>Frequent failure and severe side effects of current sarcoma therapy warrants new therapeutic approaches. The small-molecule MDM2 antagonist Nutlin-3a activates the p53 pathway and efficiently induces apoptosis in tumours with amplified <it>MDM2 </it>gene and overexpression of MDM2 protein. However, the majority of human sarcomas have normal level of MDM2 and the therapeutic potential of MDM2 antagonists in this group is still unclear. We have investigated if Nutlin-3a could be employed to augment the response to traditional therapy and/or reduce the genotoxic burden of chemotherapy.</p> <p>Methods</p> <p>A panel of sarcoma cell lines with different <it>TP53 </it>and <it>MDM2 </it>status were treated with Nutlin-3a combined with Doxorubicin, Methotrexate or Cisplatin, and their combination index determined.</p> <p>Results</p> <p>Clear synergism was observed when Doxorubicin and Nutlin-3a were combined in cell lines with wild-type <it>TP53 </it>and amplified <it>MDM2</it>, or with Methotrexate in both <it>MDM2 </it>normal and amplified sarcoma cell lines, allowing for up to tenfold reduction of cytotoxic drug dose. Interestingly, Nutlin-3a seemed to potentiate the effect of classical drugs as Doxorubicin and Cisplatin in cell lines with mutated <it>TP53</it>, but inhibited the effect of Methotrexate.</p> <p>Conclusion</p> <p>The use of Nutlin in combination with classical sarcoma chemotherapy shows promising preclinical potential, but since clear biomarkers are still lacking, clinical trials should be followed up with detailed tumour profiling.</p

Predominant Functional Expression of Kv1.3 by Activated Microglia of the Hippocampus after Status epilepticus

BACKGROUND:Growing evidence indicates that the functional state of microglial cells differs according to the pathological conditions that trigger their activation. In particular, activated microglial cells can express sets of Kv subunits which sustain delayed rectifying potassium currents (Kdr) and modulate differently microglia proliferation and ability to release mediators. We recently reported that hippocampal microglia is in a particular activation state after a status epilepticus (SE) and the present study aimed at identifying which of the Kv channels are functionally expressed by microglia in this model. METHODOLOGY/PRINCIPAL FINDINGS:SE was induced by systemic injection of kainate in CX3CR1(eGFP/+) mice and whole cell recordings of fluorescent microglia were performed in acute hippocampal slices prepared 48 h after SE. Microglia expressed Kdr currents which were characterized by a potential of half-maximal activation near -25 mV, prominent steady-state and cumulative inactivations. Kdr currents were almost abolished by the broad spectrum antagonist 4-Aminopyridine (1 mM). In contrast, tetraethylammonium (TEA) at a concentration of 1 mM, known to block Kv3.1, Kv1.1 and 1.2 subunits, only weakly reduced Kdr currents. However, at a concentration of 5 mM which should also affect Kv1.3 and 1.6, TEA inhibited about 30% of the Kdr conductance. Alpha-dendrotoxin, which selectively inhibits Kv1.1, 1.2 and 1.6, reduced only weakly Kdr currents, indicating that channels formed by homomeric assemblies of these subunits are not important contributors of Kdr currents. Finally, agitoxin-2 and margatoxin strongly inhibited the current. CONCLUSIONS/SIGNIFICANCE:These results indicate that Kv1.3 containing channels predominantly determined Kdr currents in activated microglia after SE