High-throughput proteomics of breast carcinoma cells: a focus on FTICR-MS

Discovery of better biomarkers for diagnosis, prognosis and therapy-response prediction is the most critical task of a scientific quest aimed at developing novel, tailormade therapies for patients with cancer. Consequently, a proteome-wide analysis, in addition to genomic studies, is an absolute requirement for a complete functional understanding of tumor biology. Ultra-sensitive, high-performance Fourier-transform ion-cyclotron resonance (FTICR) mass spectrometry (MS) currently holds an important role in fulfilling the demands of biomarker discovery. In this review, we describe the applicability of FTICR-MS for breast cancer proteomics, particularly for the analysis of complex protein mixtures obtained from a limited number of cells typically available from clinical specimens.     

Kir4.1-mediated spatial buffering of K+: Experimental challenges in determination of its temporal and quantitative contribution to K+ clearance in the brain

Neuronal activity results in release of K⁺ into the extracellular space of the central nervous system. If the excess K⁺ is allowed to accumulate, neuronal firing will be compromised by the ensuing neuronal membrane depolarization. The surrounding glial cells are involved in clearing K⁺ from the extracellular space by molecular mechanism(s), the identity of which have been a matter of controversy for over half a century. Kir4.1-mediated spatial buffering of K⁺ has been promoted as a major contributor to K⁺ removal although its quantitative and temporal contribution has remained undefined. We discuss the biophysical and experimental challenges regarding determination of the contribution of Kir4.1 to extracellular K⁺ management during neuronal activity. It is concluded that 1) the geometry of the experimental preparation is crucial for detection of Kir4.1-mediated spatial buffering and 2) Kir4.1 enacts spatial buffering of K⁺ during but not after neuronal activity.     

Allosteric regulation of pentameric ligand-gated ion channels: An emerging mechanistic perspective

Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger—a neurotransmitter—into an ion flux through the postsynaptic membrane. They are oligomeric assemblies that provide prototypical examples of allosterically regulated integral membrane proteins. Here, we present an overview of the most recent advances on the signal transduction mechanism based on the X-ray structures of both prokaryotic and invertebrate eukaryotic pLGICs and on atomistic Molecular Dynamics simulations. The present results suggest that ion gating involves a large structural reorganization of the molecule mediated by two distinct quaternary transitions, a global twisting and the blooming of the extracellular domain, which can be modulated by ligand binding at the topographically distinct orthosteric and allosteric sites. The emerging model of gating is consistent with a wealth of functional studies and will boost the development of novel pharmacological strategies.     

Modeling interactions between voltage-gated Ca2+ channels and KCa1.1 channels

High voltage-activated (HVA) Cav channels form complexes with KCa1.1 channels, allowing reliable activation of KCa1.1 current through a nanodomain interaction. We recently found that low voltage-activated Cav3 calcium channels also create KCa1.1-Cav3 complexes. While coimmunoprecipitation studies again supported a nanodomain interaction, the sensitivity to calcium chelating agents was instead consistent with a microdomain interaction. A computational model of the KCa1.1-Cav3 complex suggested that multiple Cav3 channels were necessary to activate KCa1.1 channels, potentially causing the KCa1.1-Cav3 complex to be more susceptible to calcium chelators. Here, we expanded the model and compared it to a KCa1.1-Cav2.2 model to examine the role of Cav channel conductance and kinetics on KCa1.1 activation. As found for direct recordings, the voltage-dependent and kinetic properties of Cav3 channels were reflected in the activation of KCa1.1 current, including transient activation from lower voltages than other KCa1.1-Cav complexes. Substantial activation of KCa1.1 channels required the concerted activity of several Cav3.2 channels. Combined with the effect of EGTA, these results suggest that the Ca²⁺ domains of several KCa1.1-Cav3 complexes need to cooperate to generate sufficient [Ca²⁺]_i, despite the physical association between KCa1.1 and Cav3 channels. By comparison, Cav2.2 channels were twice as effective at activating KCa1.1 channels and a single KCa1.1-Cav2.2 complex would be self-sufficient. However, even though Cav3 channels generate small, transient currents, the regulation of KCa1.1 activity by Cav3 channels is possible if multiple complexes cooperate through microdomain interactions.     

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca²⁺ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).¹ Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca²⁺ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca²⁺-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 μM Ca²⁺. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca²⁺ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca²⁺ and Ca²⁺ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca²⁺ transient measurements, performed in these isolated myocytes.     

Carbon Coated ZnFe2O4 Nanoparticles for Advanced Lithium‐Ion Anodes

The preparation and electrochemical characterization of a new material consisting of carbon coated ZnFe₂O₄ nanoparticles is presented. This material, which offers an interesting combination of alloying and conversion mechanisms, is capable of hosting up to nine equivalents of lithium per unit formula, corresponding to an exceptional specific capacity, higher than 1000 mAh g^?1. Composite electrodes of such a material, prepared using environmentally friendly sodium carboxymethyl cellulose as binder, showed the highest, ever reported, specific capacity and high rate performance upon long‐term testing. Furthermore, in situ X‐ray diffraction analysis allowed identifying the reduction process occurring upon initial lithiation.     

Sodium Storage and Transport Properties in Layered Na2Ti3O7 for Room‐Temperature Sodium‐Ion Batteries

Layered sodium titanium oxide, Na₂Ti₃O₇, is synthesized by a solid‐state reaction method as a potential anode for sodium‐ion batteries. Through optimization of the electrolyte and binder, the microsized Na₂Ti₃O₇ electrode delivers a reversible capacity of 188 mA h g^?1 in 1 M NaFSI/PC electrolyte at a current rate of 0.1C in a voltage range of 0.0–3.0 V, with sodium alginate as binder. The average Na storage voltage plateau is found at ca. 0.3 V vs. Na⁺/Na, in good agreement with a first‐principles prediction of 0.35 V. The Na storage properties in Na₂Ti₃O₇ are investigated from thermodynamic and kinetic aspects. By reducing particle size, the nanosized Na₂Ti₃O₇ exhibits much higher capacity, but still with unsatisfied cyclic properties. The solid‐state interphase layer on Na₂Ti₃O₇ electrode is analyzed. A zero‐current overpotential related to thermodynamic factors is observed for both nano‐ and microsized Na₂Ti₃O₇. The electronic structure, Na⁺ ion transport and conductivity are investigated by the combination of first‐principles calculation and electrochemical characterizations. On the basis of the vacancy‐hopping mechanism, a quasi‐3D energy favorable trajectory is proposed for Na₂Ti₃O₇. The Na⁺ ions diffuse between the TiO₆ octahedron layers with pretty low activation energy of 0.186 eV.