A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving

The epithelial Na+ channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na+ transport, mechanotransduction, and neurotransmission. The role of ENaC in Na+ homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC β and γ subunits causing hypertension. The function of ENaC in Na+ reabsorption depends critically on its ability to discriminate between Na+ and other ions like K+ or Ca2+. ENaC is virtually impermeant to K+ ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC α subunit (αS589), which when mutated allows larger ions such as K+, Rb+, Cs+, and divalent cations to pass through the channel. The relative ion permeability of each of the αS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that αS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na+ and Li+ ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of β1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of β1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated β1 receptors show that the cytoplasmic portion of β1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified α-actinin colocalizes and redistributes with β1 receptors on ventral plasma membranes depleted of actin, implicating binding of α-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: A quantitative approach

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na+-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the α, β, and γ ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of α, β, and γ ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (β R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Long-term plasticity in interneurons of the dentate gyrus

Single interneurons influence thousands of postsynaptic principal cells, and the control of interneuronal excitability is an important regulator of the computational properties of the hippocampus. However, the mechanisms underlying long-term alterations in the input–output functions of interneurons are not fully understood. We report a mechanism of interneuronal plasticity that leads to the functional enhancement of the gain of glutamatergic inputs in the absence of long-term potentiation of the excitatory synaptic currents. Interneurons in the dentate gyrus exhibit a characteristic, limited (≈8 mV) depolarization of their resting membrane potential after high-frequency stimulation of the perforant path. The depolarization can be observed with either whole-cell or perforated patch electrodes, and it lasts in excess of 3 h. The long-term depolarization is specific to interneurons, because granule cells do not show it. The depolarization requires the activation of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and the rise of intracellular Ca2+, but not N-methyl-d-aspartate (NMDA) receptor activation. Data on the maintenance of the depolarization point to a major role for a long-term change in the rate of electrogenic Na+/K+-ATPase pump function in interneurons. As a result of the depolarization, interneurons after the tetanus respond with action potential discharges to previously subthreshold excitatory postsynaptic potentials (EPSPs), even though the EPSPs are not potentiated. These results demonstrate that the plastic nature of the interneuronal resting membrane potential underlies a unique form of long-term regulation of the gain of excitatory inputs to γ-aminobutyric acid (GABA)ergic neurons.

Tectonic control on the sedimentary record of the central Moldavidian Basin (Eastern Carpathians, Romania)

The sedimentary record of the Tarcău and Vrancea Nappes, belonging to the flysch accretionary zone of the Eastern Carpathians (Eastern Carpathian Outer Flysch), registered Cretaceous-Miocene events during the evolution of the Moldavidian Basin. Our biostratigraphic data indicate that the deposits studied are younger than previously reported. The comparison of sedimentary record studied with the Late Cretaceous-Early Miocene global eustatic curve indicates that eustatic factor played a secondary role, after the tectonic one. Four main stages of different processes influenced by tectonics are recognized in the sedimentary record: (1) Campanian-Maastrichtian-earliest Paleocene; (2) latest Ypresian-Lutetian; (3) late Chattian-earliest Aquitanian, and (4) late Aquitanian-early Burdigalian. The late Chattian- earliest Aquitanian and late Aquitanian-early Burdigalian records indicate a high tectonic influence. The first event was related to the foredeep stage of the sedimentary domain studied, and the second one to the deformation stage of the same domain. The sedimentary records of tectonic influence recognized during these stages are useful tools for geodynamic reconstructions. The stratigraphic correlation of Tarcău and Vrancea sedimentary records are used

3D geometry of growth strata in a fault-propagation fold: insights into space–time evolution of the Crevillente Fault (Abanilla-Alicante sector), Betic Cordillera, Spain

This work presents a 3D geometric model of growth strata cropping out in a fault-propagation fold associated with the Crevillente Fault (Abanilla-Alicante sector) from the Bajo Segura Basin (eastern Betic Cordillera, southern Spain). The analysis of this 3D model enables us to unravel the along-strike and along-section variations of the growth strata, providing constraints to assess the fold development, and hence, the fault kinematic evolution in space and time. We postulate that the observed along-strike dip variations are related to lateral variation in fault displacement. Along-section variations of the progressive unconformity opening angles indicate greater fault slip in the upper Tortonian–Messinian time span; from the Messinian on, quantitative analysis of the unconformity indicate a constant or lower tectonic activity of the Crevillente Fault (Abanilla-Alicante sector); the minor abundance of striated pebbles in the Pliocene-Quaternary units could be interpreted as a decrease in the stress magnitude and consequently in the tectonic activity of the fault. At a regional scale, comparison of the growth successions cropping out in the northern and southern limits of the Bajo Segura Basin points to a southward migration of deformation in the basin. This means that the Bajo Segura Fault became active after the Crevillente Fault (Abanilla-Alicante sector), for which activity on the latter was probably decreasing according to our data. Consequently, we propose that the seismic hazard at the northern limit of the Bajo Segura Basin should be lower than at the southern limit.

The New EU Foreign Policy Architecture: Reviewing the first two years of the EEAS. CEPS Paperbacks. February 2013

This CEPS book examines two interrelated questions: 1) How has the European External Action Service (EEAS) functioned in the EU institutional architecture in the first two years of its existence? 2) What improvements can be made through the 2013 review and the 2014 revision of the EEAS’ mandate? The study contributes to the current debate through an in-depth examination of the EEAS’ relations with the EU member states, the European Commission, the European Parliament and its Delegations. The analysis is complemented by in-depth interviews conducted with senior officials from the relevant institutions. The authors put forward specific recommendations, organised around three basic roles that the EEAS plays in the EU’s external relations: a) leader, b) coordinator and c) information hub.

State of the Union and key challenges for Europe's future. CHALLENGE EUROPE Issue 22 - Challenges and new beginnings: Priorities for the EU's new leadership, September 2014.

In the last five years deep cracks have appeared in the European project. The 'euro-area crisis' triggered by a severe global financial and economic crisis has put European integration to a major test, more profound than ever before. The experience of recent years has revealed and exacerbated significant deficiencies in the European Union's (EU) economic and political construction. At time it has cast doubt on fundamentals of the European project and raised questions about whether Europe will be able to deal effectively not only with the immediate crisis, but also with the many other serious socio-economic, politico-institutional, societal and global challenges that Europe is and will be confronted with. At the start of a new institutional-political cycle (2014-2019) and while the crisis situation has for a number of reasons improved significantly since the summer of 2012, at least in systemic terms, the Union's new leadership and Member States will now have to take strategic decisions about the future of European integration.