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Molecular Characterization of the Mitochondrial Presequence TranslocaseDenkert, Niels 24 November 2017 (has links)
No description available.
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Ion selectivity and membrane potential effects of two scorpion pore-forming peptides / D. ElgarElgar, Dale January 2005 (has links)
Parabutoporin (PP) and opistoporin 1 (OP1) are cation, a-helical antimicrobial peptides isolated
from the southern African scorpion species, Parabuthus schlechteri and Opistophthalmus carinatus,
respectively. Along with their antimicrobial action against bacteria and fungi, these peptides show
pore-forming properties in the membranes of mammalian cells. Pore-formation and ion selectivity in
cardiac myocytes were investigated by measuring the whole cell leak current by means of the patch
clamp technique. Pore-formation was observed as the induction of leak currents. Ion selectivity of
the pores was indicated by the shift of the reversal potential (E,,,) upon substitution of intra (K' with
CS' and CI- with aspartate) and extracellular (Na' with NMDG') ions. Results were compared with
the effect of gramicidin A used as a positive control for monovalent cation selective pores. PP and
OP I induced a fluctuating leak current and indicate non-selectivity of PP and OP1-induced pores.
An osmotic protection assay to determine estimated pore size was performed on the cardiac
myocytes. PP and OP1-induced pores had an estimate pore size of 1.38-1.78 nm in diameter.
The effect of PP and OP1 on the membrane potential (MP) of a neuroblastoma cell line and cardiac
myocytes was investigated. TMRM was used to mark the MP fluorescently and a confocal
microscope used to record the data digitally. The resting membrane potential (RMP) of the
neuroblastoma cells was calculated at -38.3 f 1.9 mV. PP (0.5 uM) and OP1 (0.5-1 uM) depolarized
the entire cell uniformly to a MP of -1 1.9 k 3.9 mV and -9.4 k 1.9 mV, respectively. This occurred
after 20-30 min of peptide exposure. In the case of the cardiac myocytes depolarization was induced
to -39.7 f 8.4 mV and -32.6 f 5.2 mV by 0.5-1 uM PP and 1.5-2.5 uM OPl, respectively. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2006.
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Ion selectivity and membrane potential effects of two scorpion pore-forming peptides / D. ElgarElgar, Dale January 2005 (has links)
Parabutoporin (PP) and opistoporin 1 (OP1) are cation, a-helical antimicrobial peptides isolated
from the southern African scorpion species, Parabuthus schlechteri and Opistophthalmus carinatus,
respectively. Along with their antimicrobial action against bacteria and fungi, these peptides show
pore-forming properties in the membranes of mammalian cells. Pore-formation and ion selectivity in
cardiac myocytes were investigated by measuring the whole cell leak current by means of the patch
clamp technique. Pore-formation was observed as the induction of leak currents. Ion selectivity of
the pores was indicated by the shift of the reversal potential (E,,,) upon substitution of intra (K' with
CS' and CI- with aspartate) and extracellular (Na' with NMDG') ions. Results were compared with
the effect of gramicidin A used as a positive control for monovalent cation selective pores. PP and
OP I induced a fluctuating leak current and indicate non-selectivity of PP and OP1-induced pores.
An osmotic protection assay to determine estimated pore size was performed on the cardiac
myocytes. PP and OP1-induced pores had an estimate pore size of 1.38-1.78 nm in diameter.
The effect of PP and OP1 on the membrane potential (MP) of a neuroblastoma cell line and cardiac
myocytes was investigated. TMRM was used to mark the MP fluorescently and a confocal
microscope used to record the data digitally. The resting membrane potential (RMP) of the
neuroblastoma cells was calculated at -38.3 f 1.9 mV. PP (0.5 uM) and OP1 (0.5-1 uM) depolarized
the entire cell uniformly to a MP of -1 1.9 k 3.9 mV and -9.4 k 1.9 mV, respectively. This occurred
after 20-30 min of peptide exposure. In the case of the cardiac myocytes depolarization was induced
to -39.7 f 8.4 mV and -32.6 f 5.2 mV by 0.5-1 uM PP and 1.5-2.5 uM OPl, respectively. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2006.
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Modelování interakce proteinů a peptidů s kovovými ionty / Modelling of the interaction of proteins and peptides with metal ionsGutten, Ondrej January 2010 (has links)
Modelling of interactions of proteins and peptides with metal ions Ondrej Gutten - Diploma thesis Keywords: Metalloproteins, metal ion selectivity, in silico prediction Abstract: An approach for in silico prediction and estimation of selectivity properties of metal-binding peptides is suggested. An in-depth analysis is performed to disclose the justifiability and limitations of this approach. The study is divided into three parts. First part investigates the soundness of two quantum chemical methods (MP2 and DFT) for their use in the set-up quest. The testing includes comparison with CCSD(T), effect of basis selection, performance of the two methods in geometry optimizations and effect of implicit solvent model. Second part foreshadows the approach of searching for a metal selective peptide by thoroughly investigating the ability of simple representative systems, derived from their metalloprotein templates, to retain the property of interest. Final part describes the initial step of extensive combinatorial approach towards examination of vast number of simple systems that represent metal-binding sites, and which are to be used for prediction of metal-selectivity through exploitation of the described approach and, ultimately, to the de novo design of metalloproteins with desired properties.
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Electrophysiological characterization of the human two-pore channel 2Lam, Andy Ka Ming January 2015 (has links)
The Two-pore channel (TPC1-3) family represents a recently identified class of endolysosomal ion channels. TPCs were originally proposed to be promising candidate channels for NAADP-induced Ca<sup>2+</sup> release. However, subsequent studies have emerged to propose an alternative view where TPCs may be Na+-selective channels regulated by the lysosome-specific phosphoinositide PI(3,5)P2 or voltage in an isoform-dependent manner. This thesis asks the question of whether pharmacological and ion permeation properties of TPCs, in particular the human TPC2, may satisfy or may be consistent with the requirement of a potential NAADP-sensitive Ca<sup>2+</sup>-release channel. These fundamental properties of hTPC2 were approached using patch-clamp electrophysiology and confocal fluorescence microscopy, and were analysed quantitatively to extract relevant physical parameters important to our understanding of their physiological and functional significance. Chapter 2 presents the basic electrophysiological characterisation of hTPC2. It follows a logical way by first determining the ion permeation properties, followed by the investigation of its physical relation with fractional Ca<sup>2+</sup> current and Ca<sup>2+</sup> nanodomains to rigorously prove that this Na<sup>+</sup> selectivity is sufficient to ensure negligible Ca<sup>2+</sup> leakage both experimentally and theoretically. This follows the logic that matter must not be created nor destroyed so that a Na+-selective channel that poses a physiologically significant energy barrier to Ca<sup>2+</sup> permeation from one side would not lead to the creation of Ca<sup>2+</sup> on the other side. Chapter 3 represents a natural progression from Chapter 2 and is aimed at investigating the underlying mechanisms responsible for the electrophysiological ion selectivity observed. This chapter also follows a logical way by first identifying spermine as a high valence intracellular blocker, its mutual antagonism with different external ionic species that allows the determination of ion-binding affinity, followed by the determination of the concentration dependence of ion conduction to identify possible lower affinity binding. By considering all the above qualities, the outcome is a coherent description and connection of ion binding selectivity, kinetic selectivity and ion binding configuration with the observed electrophysiological selectivity. Chapter 4 discusses the missing puzzles and how these questions might be addressed.
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Development of A Cryogenic Drift Cell Spectrometer and Methods for Improving the Analytical Figures of Merit for Ion Mobility-Mass Spectrometry AnalysisMay, Jody C. 2009 August 1900 (has links)
A cryogenic (325-80 K) ion mobility-mass spectrometer was designed and
constructed in order to improve the analytical figures-of-merit for the chemical analysis
of small mass analytes using ion mobility-mass spectrometry. The instrument
incorporates an electron ionization source, a quadrupole mass spectrometer, a uniform
field drift cell spectrometer encased in a cryogenic envelope, and an orthogonal
geometry time-of-flight mass spectrometer. The analytical benefits of low temperature
ion mobility are discussed in terms of enhanced separation ability, ion selectivity and
sensitivity. The distinction between resolving power and resolution for ion mobility is
also discussed. Detailed experimental designs and rationales are provided for each
instrument component. Tuning and calibration data and methods are also provided for
the technique.
Proof-of-concept experiments for an array of analytes including rare gases
(argon, krypton, xenon), hydrocarbons (acetone, ethylene glycol, methanol), and halides
(carbon tetrachloride) are provided in order to demonstrate the advantages and limitations of the instrument for obtaining analytically useful information. Trendline
partitioning of small analyte ions based on chemical composition is demonstrated as a
novel chemical analysis method. The utility of mobility-mass analysis for mass selected
ions is also demonstrated, particularly for probing the ion chemistry which occurs in the
drift tube for small mass ions.
As a final demonstration of the separation abilities of the instrument, the
electronic states of chromium and titanium (ground and excited) are separated with low
temperature. The transition metal electronic state separations demonstrated here are at
the highest resolution ever obtained for ion mobility methods. The electronic
conformational mass isomers of methanol (conventional and distonic) are also partially
separated at low temperature. Various drift gases (helium, neon, and argon) are explored
for the methanol system in order to probe stronger ion-neutral interaction potentials and
effectuate higher resolution separations of the two isomeric ions. Finally, two versatile
ion source designs and a method for axially focusing ions at low pressure (1-10 torr)
using electrostatic fields is presented along with some preliminary work on the ion
sources.
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Biophysical Studies On The Plastic And Cooperative Properties Of Single Voltage Gated Na+ And Leak K+ Ion ChannelsNayak, Tapan Kumar 11 1900 (has links)
Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. There are mounting evidences suggesting that the kinetic properties of ion channels undergo activity-dependent changes in various pathophysiological conditions. Here such activity-dependent changes were studied in case of two different ion channels; the rat brain derived voltage-gated Na+ channel, rNav1.2 and the human background leak K+ channel, hTREK1 using the single channel patch-clamp technique. Our results on the voltage-gated Na+ channel (Chapter III) illustrated that sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, induced a defined non-linear variation in the unitary conductance, activation, inactivation and recovery kinetic properties of the channel. Signal processing tools attributed a pseudo-oscillatory nature to the non-linearity observed in the channel properties. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clustering of dwell time events and strong autocorrelation in the dwell time series. The persistence of such molecular memory was found to be dependent on the duration of depolarization.
Similar plastic changes were observed in case of the hTREK1 channel in presence of saturating concentrations of agonist, trichloroethanol (TCE) (Chapter IV). TREK1 channel behaves similar to single enzyme molecules with a single binding site for the substrate K+ ion whereas TCE acts as an allosteric activator of the channel. We observed that with increasing concentration of TCE (10 M to 10 mM) the catalytic turnover rate exhibited progressive departure from monoexponential to multi-exponential distribution suggesting the presence of ‘dynamic disorder’ analogous to single enzyme molecules. In addition, we observed the induction of strong correlation in successive waiting times and flux intensities, exemplified by distinct mode switching between high and low flux activity, which implied the induction of memory in single ion channel. Our observation of such molecular memory in two different ion channels in different experimental conditions highlights the importance and generality of the phenomenon which is normally hidden under the ensemble behaviour of ion channels. In the final part of the work (chapter V) we observed strong negative cooperativity and half-of-sites saturation kinetics in the interaction of local anesthetic, lidocaine with hTREK1 channel. We also mapped the specific anesthetic binding site in the c-terminal domain of the channel. Further, single channel analysis and the heterodimer studies enabled us to propose a model for this interaction and provide a plausible paradigm for the inhibitory action of lidocaine on hTREK1.
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Ion selectivity in carrier-mediated dialysis and electrodialysisHansen, Steven Paul 02 May 2012 (has links)
Membrane transport processes underlie many purification technologies. The efficiency of a membrane separation process depends upon material throughput (flux), and the degree to which the membrane discriminates amongst species in the feed stock (selectivity). In a supported liquid membrane, flux may be enhanced by carrier molecules, which act as catalysts of translocation. Carrier molecules also confer selectivity, via differential molecular recognition of the substances in the feed stock. The effect of electrical potential on the flux and selectivity of carrier-containing supported liquid membranes is not well documented. We elected to study the effect of electrical potential on supported liquid membranes containing valinomycin, a potassium ionophore, and a calixarene ester, a sodium ionophore. In these systems, the open circuit membrane potential could be made positive or negative by the choice of anion. With both of these carriers, we observed that selectivity for potassium or sodium salts was dependent on the open circuit membrane potential. To confirm that electrical potential was responsible for the observed selectivity variance, we applied a potential across the membrane using a potentiostat. The applied potential created conditions for carrier-mediated electrodialysis, where oxidation and reduction reactions on either side of the membrane act as the driving force for transmembrane flux of charged species. In chronoamperometry experiments, we found that selectivity for potassium or sodium ion was dependent on the applied electrical potential. Subject to some constraints, selectivity and flux could be controlled by the application of positive or negative electrical potentials. Linear sweep voltammetry experiments allowed for the rapid prediction of the potential that must be applied to achieve optimal selectivity. We also found that membrane potential measurements, as well as the magnitude of current that flows in chronoamperometry experiments, could be interpreted to predict Eisenman and Hofmeister sequences. These results are novel, and await a convincing theoretical justification. The results also suggest that a separation technology could be developed around the idea of modulating selectivity with electrical potential. In this regard, carrier-mediated electrodialysis may be suitable for the sequestration of toxic or radioactive heavy metals, and a large number of carrier molecules for metal ions are currently known. The technique may also be suitable for separating organic molecules, such as high-value chiral pharmaceuticals. Supported liquid membranes are a useful research tool, but industrial applications may require a more stable membrane architecture. / Graduate
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Structural dynamics of the selectivity filter in HCN1 ion channelAhrari, Sajjad 05 1900 (has links)
Les canaux HCN (cycliques nucléotidiques) activés par hyperpolarisation appartiennent à la superfamille des canaux cationiques voltage-dépendants et sont responsables de la génération de courant drôle (If) dans les cellules cardiaques et neuronales. Malgré la similitude structurelle globale avec le potassium voltage-dépendant (Kv) et les canaux ioniques cycliques nucléotidiques (CNG), ils montrent un modèle de sélectivité distinctif pour les ions K+ et Na+. Plus précisément, leur perméabilité accrue aux ions Na+ est essentielle à son rôle dans la dépolarisation des membranes cellulaires. Ils sont également l'une des seules protéines connues à sélectionner entre les ions Na+ et Li+, faisant des HCN des canaux semi-sélectifs. Ici, nous étudions les propriétés de sélectivité uniques des canaux HCN à l'aide de simulations de dynamique moléculaire. Nos simulations suggèrent que le pore HCN1 est très flexible et dilaté par rapport aux canaux Kv et qu'il n'y a qu'un seul site de liaison ionique stable dans le filtre de sélectivité qui les distingue des canaux Kv et CNG. Nous observons également que la coordination et l'hydratation des ions diffèrent dans le filtre de sélectivité de HCN1 par rapport aux canaux Kv et CNG. De plus, la coordination des ions K+ par les groupes carbonyle du filtre de sélectivité est plus stable par rapport aux ions Na+ et Li+, ce qui peut expliquer les propriétés de sélectivité distinctes du canal. / Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the voltage-gated cation channel superfamily and are responsible for the generation of funny current (If) in cardiac and neuronal cells. Despite the overall structural similarity to voltage-gated potassium (Kv) and cyclic nucleotide-gated (CNG) ion channels, they show distinctive selectivity pattern for K+ and Na+ ions. Specifically, their increased permeability to Na+ ions is critical to its role in depolarizing cellular membranes. They are also one of the only known proteins to select between Na+ and Li+ ions, making HCNs semi-selective channels. Here we investigate the unique selectivity properties of HCN channels using molecular dynamics simulations. Our simulations suggest that the HCN1 pore is very flexible and dilatated compared to Kv channels and that there is only one stable ion binding site within the selectivity filter which discriminates them from both Kv and CNG channels. We also observe that ion co-ordination and hydration differ within the selectivity filter of HCN1 compared to Kv and CNG channels. Additionally, the co-ordination of K+ ions by the carbonyl groups of the selectivity filter is more stable compared to Na+ and Li+ ions, which may explain the channel's distinct selectivity properties.
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Structural analysis of ion permeation in a non-selective channel mimicking the AMPA receptorMinniberger, Sonja 08 December 2021 (has links)
Ionenkanäle spielen eine wichtige Rolle in vielen physiologischen Prozessen. Während manche Kanäle hochspezifisch für eine Ionensorte sind, sind andere Kanäle weniger selektiv. Tetramere Ionenkanäle weisen eine gemeinsame Grundarchitektur auf, von der nur ionotrope Glutamatrezeptoren (iGluRs) abweichen, deren Struktur im Vergleich zu den anderen invertiert ist. Eine Untergruppe der iGluRs stellen α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Rezeptoren (AMPARs) dar, welche im Zentralnervensystem von Wirbeltieren die Mehrheit der exzitatorischen Neurotransmission übernehmen. Eine einzelne posttranskriptionelle Veränderung (Glutamin zu Arginin) an der Spitze des Selektivitätsfilters (SF, Q/R Stelle) von AMPAR-Typ 2 (GluA2) macht den Kanal quasi undurchlässig für Ca2+.
Das Ziel dieser Arbeit war das Erstellen einer GluA2-Kanalchimäre mit Hilfe des bakteriellen Kanals NaK. Mutation rund um die Q/R Stelle wurden nicht toleriert von der Chimäre, während C-terminale Mutationen des SF stabil waren und für strukturelle Studien verwendet wurden. Die Entfernung einer Aminosäure im Vergleich zum NaK Wildtyp erzeugte eine Begradigung des SF und eine Erweiterung der wassergefüllten Ausbuchtung. Überraschenderweise kristallisierten die NaK Chimären überwiegend in zweifach symmetrischer Anordnung. Vor allem im SF kam es zu ausgeprägter lokaler Asymmetrie, unabhängig von der Ionenzusammensetzung. Um die Flexibilität des SF näher zu untersuchen, wurden Aminosäuren von NaK in GluA2 integriert und mithilfe von Patch-clamp Elektrophysiologie untersucht. Gleichsam wie in NaK, wurden Mutationen an der Filterspitze nicht toleriert, wohingegen die C-terminale Hälfte problemlos ausgetauscht werden konnte. Die funktionelle Integrität der NaK Chimäre wurde mithilfe von Einzelkanalmessungen in Bilayern überprüft. Zusätzlich wurden, auf Basis der gelösten Strukturen, Molekulardynamiksimulationen durchgeführt, welche einen dynamischen Einblick in den Permeationsmechanismus erlauben. / Ion channels play an important role in many physiological processes, for example the generation of action potentials. While some channels display high selectivity for one ion species, others are more promiscuous. All tetrameric cation channels share the same principal architecture, but the transmembrane domain of ionotropic glutamate receptors (iGluRs) is inverted relative to the other members. A subfamily of iGluRs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), mediates the vast majority of excitatory neurotransmission in the vertebrate central nervous system. In AMPA-type 2 iGluRs (GluA2), a single post-transcriptional modification (glutamine to arginine) at the tip of the selectivity filter (SF, Q/R site) renders the channel Ca2+ impermeable. The aim of this thesis was therefore to create an AMPAR pore mimic by using the bacterial channel NaK. Unfortunately, mutations around the Q/R site abolished expression of the NaK-GluA2 chimera, while C-terminal mutations of the SF were stable and could be used for structural studies. The shortening of the SF by one amino acid caused a straightening and opened an extended water-filled vestibule. Strikingly, most of the tested chimeras exhibited twofold symmetry with strong local asymmetry in the mutated part of the SF independent of the ion type present. For functional tests, residues from NaK wt were also swapped into GluA2. N-terminal mutations abolished the current response, whereas C-terminal mutations behaved wt-like. Single-channel bilayer experiments confirmed the functional integrity of the NaK-GluA2 chimera. Additionally, extensive molecular dynamics simulations, based on the solved structures, were carried out alongside.
In summary, it could be demonstrated that the SF of the non-selective NaK-GluA2 chimera is highly flexible and accommodated all tested ions. Ion binding is accompanied by local asymmetric rearrangements, possibly creating an energetically simple way to allow permeation.
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