Elf magnetic field influence on ION Channels studied by Patch Clamp Technique: exposure set up and "Whole Cell" measurements on Potassium currents

The aim of this thesis was to study the effects of extremely low frequency (ELF) electromagnetic magnetic fields on potassium currents in neural cell lines ( Neuroblastoma SK-N-BE ), using the whole-cell Patch Clamp technique. Such technique is a sophisticated tool capable to investigate the electrophysiological activity at a single cell, and even at single channel level. The total potassium ion currents through the cell membrane was measured while exposing the cells to a combination of static (DC) and alternate (AC) magnetic fields according to the prediction of the so-called ‘ Ion Resonance Hypothesis ’. For this purpose we have designed and fabricated a magnetic field exposure system reaching a good compromise between magnetic field homogeneity and accessibility to the biological sample under the microscope. The magnetic field exposure system consists of three large orthogonal pairs of square coils surrounding the patch clamp set up and connected to the signal generation unit, able to generate different combinations of static and/or alternate magnetic fields. Such system was characterized in term of field distribution and uniformity through computation and direct field measurements. No statistically significant changes in the potassium ion currents through cell membrane were reveled when the cells were exposed to AC/DC magnetic field combination according to the afore mentioned ‘Ion Resonance Hypothesis’.

Numerical simulation of ion transport through ion channels and solid-state nanopores

Ion channels are pore-forming proteins that regulate the flow of ions across biological cell membranes. Ion channels are fundamental in generating and regulating the electrical activity of cells in the nervous system and the contraction of muscolar cells. Solid-state nanopores are nanometer-scale pores located in electrically insulating membranes. They can be adopted as detectors of specific molecules in electrolytic solutions. Permeation of ions from one electrolytic solution to another, through a protein channel or a synthetic pore is a process of considerable importance and realistic analysis of the main dependencies of ion current on the geometrical and compositional characteristics of these structures are highly required. The project described by this thesis is an effort to improve the understanding of ion channels by devising methods for computer simulation that can predict channel conductance from channel structure. This project describes theory, algorithms and implementation techniques used to develop a novel 3-D numerical simulator of ion channels and synthetic nanopores based on the Brownian Dynamics technique. This numerical simulator could represent a valid tool for the study of protein ion channel and synthetic nanopores, allowing to investigate at the atomic-level the complex electrostatic interactions that determine channel conductance and ion selectivity. Moreover it will provide insights on how parameters like temperature, applied voltage, and pore shape could influence ion translocation dynamics. Furthermore it will help making predictions of conductance of given channel structures and it will add information like electrostatic potential or ionic concentrations throughout the simulation domain helping the understanding of ion flow through membrane pores.

A hybrid technology for parallel recording of single ion channels

Hybrid technologies, thanks to the convergence of integrated microelectronic devices and new class of microfluidic structures could open new perspectives to the way how nanoscale events are discovered, monitored and controlled. The key point of this thesis is to evaluate the impact of such an approach into applications of ion-channel High Throughput Screening (HTS)platforms. This approach offers promising opportunities for the development of new classes of sensitive, reliable and cheap sensors. There are numerous advantages of embedding microelectronic readout structures strictly coupled to sensing elements. On the one hand the signal-to-noise-ratio is increased as a result of scaling. On the other, the readout miniaturization allows organization of sensors into arrays, increasing the capability of the platform in terms of number of acquired data, as required in the HTS approach, to improve sensing accuracy and reliabiity. However, accurate interface design is required to establish efficient communication between ionic-based and electronic-based signals. The work made in this thesis will show a first example of a complete parallel readout system with single ion channel resolution, using a compact and scalable hybrid architecture suitable to be interfaced to large array of sensors, ensuring simultaneous signal recording and smart control of the signal-to-noise ratio and bandwidth trade off. More specifically, an array of microfluidic polymer structures, hosting artificial lipid bilayers blocks where single ion channel pores are embededed, is coupled with an array of ultra-low noise current amplifiers for signal amplification and data processing. As demonstrating working example, the platform was used to acquire ultra small currents derived by single non-covalent molecular binding between alpha-hemolysin pores and beta-cyclodextrin molecules in artificial lipid membranes.

Computational investigations of ion conduction mechanisms using enhanced sampling methods

Proper ion channels’ functioning is a prerequisite for a normal cell and disorders involving ion channels, or channelopathies, underlie many human diseases. Long QT syndromes (LQTS) for example may arise from the malfunctioning of hERG channel, caused either by the binding of drugs or mutations in HERG gene. In the first part of this thesis I present a framework to investigate the mechanism of ion conduction through hERG channel. The free energy profile governing the elementary steps of ion translocation in the pore was computed by means of umbrella sampling simulations. Compared to previous studies, we detected a different dynamic behavior: according to our data hERG is more likely to mediate a conduction mechanism which has been referred to as “single-vacancy-like” by Roux and coworkers (2001), rather then a “knock-on” mechanism. The same protocol was applied to a model of hERG presenting the Gly628Ser mutation, found to be cause of congenital LQTS. The results provided interesting insights about the reason of the malfunctioning of the mutant channel. Since they have critical functions in viruses’ life cycle, viral ion channels, such as M2 proton channel, are considered attractive targets for antiviral therapy. A deep knowledge of the mechanisms that the virus employs to survive in the host cell is of primary importance in the identification of new antiviral strategies. In the second part of this thesis I shed light on the role that M2 plays in the control of electrical potential inside the virus, being the charge equilibration a condition required to allow proton influx. The ion conduction through M2 was simulated using metadynamics technique. Based on our results we suggest that a potential anion-mediated cation-proton exchange, as well as a direct anion-proton exchange could both contribute to explain the activity of the M2 channel.

Ion channels in mixed tethered bilayer lipid membranes

Mixed tethered bilayer lipid membranes (tBLMs) are described based on the self-assembly of a monolayer on template stripped gold, of an archea analogue thiolipid, 2,3-di-o-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L--lipoic acid ester lipid (DPTL), and a newly designed dilution molecule, tetraethylene glycol-D,L--lipoic acid ester (TEGL). The usage of spacer and addition of extra dilution molecules between the substrate and the bilayer is that this architecture provides an ionic reservoir underneath the membrane, avoiding direct contact of the embedded membrane proteins with the gold electrodes and increasing the lateral diffusion of the bilayer, thus allowing for the incorporation of complex channels proteins which are failed in non-diluted systems. The tBLM is completed by fusion of liposomes made from a mixture of 1,2-diphythanolyl-sn-glycero-3-phosphocholine (DPhyPC), cholesterol, and 1,2-diphytanoyl-sn-Glycero-3-phosphate (DPhyPG) in a molar ratio of 6:3:1. Varying the mixing ratio, the optimum mixing ratio was obtained at a dilution factor of DPTL and TEGL at 90%:10%. Only under these conditions, the mixed tBLM showed electrical properties, as shown by EIS, which are comparable to a BLM. With higher dilution factors, a defect-free lipid bilayer was not formed. Formation of bilayers have been characterized by different techniques, such as surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and quartz crystal microbalance (QCM). Different proteins such as hemolysin, melittin, gramicidin, M2, Maxi-K, nAChR and bacteriohodopsin are incorporated into these tBLMs as shown by SPR and EIS studies. Ionic conductivity at 0 V vs. Ag|AgCl, 3M KCl were measured by EIS measurements. Our results indicate that these proteins have been successfully incorporated into a very stable tBLM environment in a functionally active form. Therefore, we conclude that the mixed tBLMs have been successfully designed as a general platform for biosensing and screening purposes of membrane proteins.

Coded slotted aloha with multipacket reception over block fading channels (for satellite networks)

Random access (RA) protocols are normally used in a satellite networks for initial terminal access and are particularly effective since no coordination is required. On the other hand, contention resolution diversity slotted Aloha (CRDSA), irregular repetition slotted Aloha (IRSA) and coded slotted Aloha (CSA) has shown to be more efficient than classic RA schemes as slotted Aloha, and can be exploited also when short packets transmissions are done over a shared medium. In particular, they relies on burst repetition and on successive interference cancellation (SIC) applied at the receiver. The SIC process can be well described using a bipartite graph representation and exploiting tools used for analyze iterative decoding. The scope of my Master Thesis has been to described the performance of such RA protocols when the Rayleigh fading is taken into account. In this context, each user has the ability to correctly decode a packet also in presence of collision and when SIC is considered this may result in multi-packet reception. Analysis of the SIC procedure under Rayleigh fading has been analytically derived for the asymptotic case (infinite frame length), helping the analysis of both throughput and packet loss rates. An upper bound of the achievable performance has been analytically obtained. It can be show that in particular channel conditions the throughput of the system can be greater than one packets per slot which is the theoretical limit of the Collision Channel case.

Ubiquitylation and SUMOylation of Cardiac Ion Channels

Cardiac ion channels play an essential role in the generation of the action potential of cardiomyocytes. Over the past 15 years, a new field of research called channelopathies has emerged; it regroups all diseases caused by ion channel dysfunction. Investigators have largely determined the physiological roles of cardiac ion channels, but little is known about the molecular determinants of their regulation. Two post-translational mechanisms that are crucial in determining the fate of proteins are ubiquitylation and the SUMOylation pathways, which lead to the degradation and/or regulation of modified proteins. Recently, several groups have investigated the physiological impacts of these mechanisms on the regulation of different classes of cardiac ion channels. The objective of this review is to summarize and briefly discuss these results.

Suicidal knife wound to the heart: challenges in reconstructing wound channels with post mortem CT and CT-angiography

We present a case of an individual who stabbed himself through the heart with a large knife. Post mortem computed tomography (CT) and CT-angiography were used to assess the stab channel and to reconstruct the sequence of events. After penetrating injuries to the chest, both the intra-thoracic organs and the injury causing instrument may shift (e.g. from pnemothorax) and render forensic reconstructions more challenging. This case report illustrates the potentials and the pitfalls of CT for the reconstruction of penetrating injures to the chest.

Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.

Channels and transporters. Mini-symposium of the Division of Medicinal Chemistry (DMC) of the Swiss Chemical Society (SCS) at the Department of Chemistry, University of Basel, May 27, 2010

During a half-day symposium, the topic 'Channels and Transporters' was covered with five lectures, including a presentation on 'Introduction and Basics of Channels and Transporters' by Beat Ernst, lectures on structure, function and physiology of channels and transporters ('The Structural Basis for Ion Conduction and Gating in Pentameric Ligand-Gated Ion Channels' by Raimund Dutzler and 'Uptake and Efflux Transporters for Endogenous Substances and for Drugs' by Dietrich Keppler), and a case study lecture on 'Avosentan' by Werner Neidhart. The program was completed by Matthias Hediger who introduced to the audience the National Center of Competence in Research (NCCR)-TransCure in his lecture entitled 'From Transport Physiology to Identification of Therapeutic Targets'.

Monepantel allosterically activates DEG-3/DES-2 channels of the gastrointestinal nematode Haemonchus contortus

Monepantel is the first drug of a new family of anthelmintics, the amino acetonitrile derivatives (AAD), presently used to treat ruminants infected with gastrointestinal nematodes such as Haemonchus contortus. Monepantel shows an excellent tolerability in mammals and is active against multidrug-resistant parasites, indicating that its molecular target is absent or inaccessible in the host and is different from those of the classic anthelmintics. Genetic approaches with mutant nematodes have suggested acetylcholine receptors of the DEG-3 subfamily as the targets of AADs, an enigmatic clade of ligand-gated ion channels that is specific to nematodes and does not occur in mammals. Here we demonstrate direct interaction of monepantel, its major active metabolite monepantel sulfone, and other AADs with potential targets of the DEG-3 subfamily of acetylcholine receptors. H. contortus DEG-3/DES-2 receptors were functionally expressed in Xenopus laevis oocytes and were found to be preferentially activated by choline, to permeate monovalent cations, and to a smaller extent, calcium ions. Although monepantel and monepantel sulfone did not activate the channels by themselves, they substantially enhanced the late currents after activation of the channels with choline, indicating that these AADs are type II positive allosteric modulators of H. contortus DEG-3/DES-2 channels. It is noteworthy that the R-enantiomer of monepantel, which is inactive as an anthelmintic, inhibited the late currents after stimulation of H. contortus DEG-3/DES-2 receptors with choline. In summary, we present the first direct evidence for interaction of AADs with DEG-3-type acetylcholine receptors and discuss these findings in the context of anthelmintic action of AADs.