Verbesserte sub-µs Schaltanalyse weist auf Konformationsänderungen hin, verursacht durch Ion-Pore-Wechselwirkungen im MaxiK-Kanal

This thesis aims at the analysis of fast gating events in ion channels. Experiments were done on single MaxiK channels expressed in HEK cells using the patch clamp technique in voltage clamp mode. Crucial for high temporal resolution was the signal-to-noise ratio which was improved by optimizing the set-up and pipette treatment. Furthermore, temporal resolution was increased by sharpening the mathematical tools employed in the analysis of the kinetic behavior of single-channel currents. This was done along four lines. 1. Splitting amplitude histograms into distributions-per-level and investigating their properties with respect to model discrimination, determination of the rate constants of fast gating and the detection of failures of jump detection. 2. Creating the SQ fit, a two-step fit algorithm consisting of a direct fit of patch clamp time series, by means of a 1-step prediction algorithm and a fit of the distribution-per-open-level by means of beta distributions. 3. Finding and testing the ability of the beta fit to determine the true single-channel current in time series where the apparent current was attenuated by averaging over undetected fast gating. 4. Comparing the SQ fit with the 2-dimensional dwell-time fit as improved by T. Huth. After having developed the new tools, they were applied to the analysis of fast gating events. Two phenomena could gain the outmost benefit from the new analytical tools: 1. The negative slope in the current-voltage curve occurring at positive potentials in symmetrical solutions with K+ as the only monovalent cation. 2. The negative slope occurring at negative potentials in luminal K+/Tl+ solution when the current dragged Tl+ into the channel. Both kinds of measurement resulted in insights about ion/protein interaction. The key for the creation of molecular models of ion/channel interactions was found in the experiments with K+ as the sole monovalent cation. In the range of the negative slope, saturation of the true single-channel current and onset of fast gating occurred in the same voltage range. This finding suggested a mechanistic model: On the cytosolic side, diffusion limitation produced saturation of outward-current. This caused ion depletion in the selectivity filter. The idea that gating occurred when permeant ions did no longer compensate the repulsive forces of the carbonyl groups was supported by MD simulations reported in the literature. The model correctly predicted the effect of different K+ concentrations. For explaining the negative slope induced by negative membrane potentials when luminal Tl+ was dragged into the channel, the basic model had to be extended. Here, the effect of luminal diffusion limitation on voltage-induced gating was not sufficient for explaining the experimental findings. This effect had to be modulated by additional Tl+ binding sites in the cavity or at the inner side of the selectivity filter. Describing the full set of gating phenomena induced by Tl+ would require the analysis of the “Great Markov Model” of the MaxiK channel. It is shown by preliminary approaches with classical tools what kind of insights would be obtained from this kind of analysis and what kind of problems keep the ratio “scientific output/time” at a very low level. Nevertheless, examples are given that the analysis of beta distributions can be helpful for the extension of the Markov model. Especially the picture of a rattly channel has arisen, implying that open states are not really open and closed states are not really closed. Finally, it is discussed that using the viral channel Kcv instead of MaxiK offers better chances to continue the building of a bridge between electrophysiology and structural biology as has been initiated in this thesis.Die vorliegende Arbeit befasst sich mit der Analyse schneller Schaltprozesse in einzelnen Ionenkanälen. Die Patch-Clamp Experimente wurden an mit humanen MaxiK-Kanälen stabil transfizierten HEK-Zellen vorgenommen. Wichtig zur Erzielung einer guten Zeitauflösung ist das Signal-Rausch-Verhältnis. Dazu diente die Verbesserung des Messaufbaus und der Patchpipetten. Entscheidender für die Verbesserung der Zeitauflösung war hier jedoch die Weiterentwicklung der Programme zur kinetischen Analyse von Einzelkanalaufzeichnungen. Die vier Kernpunkte hierbei waren folgende: 1. Aufspaltung der Amplitudenhistogramme in Einzelhistogramme. Diese so genannten „distributions-per-level“ erwiesen sich als nützlich in den Bereichen der Modellunterscheidung, der Bestimmung schneller Ratenkonstanten und der Erkennung von Fehlern in der Sprungdetektion. 2. Die Entwicklung eines Programms, das zwei Fitverfahren miteinander kombiniert (SQ-Fit): Den direkten Fit der Zeitreihe mit Einschrittprädiktion und den Fit der Amplitudenhistogramme mit Betaverteilungen. 3. Entwicklung und Erprobung einer neuen Anwendung für Betaverteilungen zur Rekonstruktion des wahren Einzelkanalstroms in Aufzeichnungen, in denen der scheinbare Strom durch nicht aufgelöstes schnelles Schalten stark reduziert ist. 4. Vergleich der Leistungsfähigkeit des SQ-Fits mit dem von Tobias Huth weiterentwickelten Fit der zweidimensionalen Dwell-Time-Histogramme. Die neuen Werkzeuge wurden zur Analyse schneller Schaltereignisse eingesetzt. Zwei Phänomene waren dabei besonders interessant, weil sie wichtige Hinweise zur Ionen-Protein-Interaktion gaben: 1. Die negative Kennlinie bei positiven Membranpotentialen in der Stromspannungskurve in symmetrischen Messlösungen mit K+ als einzigem einwertigen Kation. 2. Die negative Kennlinie bei negativen Membranpotentialen wenn eine K+/Tl+- Mischung von der luminalen Seite in die Pore gezogen wird. Der Schlüssel zur Modellbildung für die Ionen-Protein-Interaktion fand sich in den Kalium-Experimenten: Im Bereich der negativen Kennlinie setzten das schnelle Schalten und die Sättigung des wahren Einzelkanalstroms ungefähr gleichzeitig ein. Dies führte zu folgendem Modell: Eine Diffusionslimitierung am cytosolischen Porenmund bewirkt eine Sättigung des Auswärtsstromes und eine Verarmung an Kaliumionen im Selektivitätsfilter. Letzteres destabilisiert das Filter und führt zu Schließungen, weil die abstoßenden Kräfte der sich gegenüberliegenden Karbonylgruppen nicht mehr kompensiert werden. MD-Simulationen in der Literatur unterstützen diese Annahme. Das Modell war in der Lage, die Spannungs- und Konzentrationsabhängigkeit der beobachteten Effekte vorherzusagen. Zur Erklärung der Tl+-induzierten negativen Kennlinie bei negativen Spannungen musste das Modell erweitert werden, da die Annahme ähnlicher Diffusionslimitierungen am luminalen Porenmund nicht ausreichend war, um die Messergebnisse zu erklären. Es wurde vermutet, dass es in der Cavity oder am inneren Ende des Selektivitätsfilters Tl+-Bindungsstellen gibt, die das Schalten zusätzlich modulieren. Das gesamte Schaltverhalten der Kanäle mit und ohne Tl+ zu beschreiben, erfordert ein wesentlich größeres Markov-Modell als jenes mit nur zwei Zuständen, das zur Analyse des schnellen Schalten in den Bursts benutzt wurde. Ein rein mathematisches Vorgehen, um dieses Modell zu finden, würde unverhältnismäßig viel Zeit erfordern. Darum wurde die Analyse nur kurz angerissen, um aufzuzeigen, in welche Richtung es gehen kann. Dabei zeigte sich, dass die Analyse von Betaverteilungen wichtige Beiträge zur Modellentwicklung liefern kann: So entstand das Bild des „klapprigen“ Kanals, bei dem die Offenzustände nicht richtig offen und die Geschlossenzustände nicht richtig geschlossen sind. Zum Schluss wird noch diskutiert, dass ein Wechsel des Untersuchungsobjektes vom MaxiK zum wesentlich einfacheren viralen Kcv in zukünftigen Arbeiten vermutlich die hier begonnene Brückenbildung zwischen Elektrophysiologie und Strukturbiologie besser fördern wird

Nickel as a co-catalyst for photocatalytic hydrogen evolution on graphitic-carbon nitride (sg-CN): what is the nature of the active species?

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.The nature of a nickel-based co-catalyst deposited on a sol-gel prepared porous graphitic-carbon nitride (sg-CN), for photocatalytic H-2 production from water, has been investigated. The formation of the active catalytic species, charge separation and recombination of the photogenerated electrons and holes during photochemical H-2 evolution has been determined for the first time using in situ EPR spectroscopy.DFG, EXC 314, Unifying Concepts in CatalysisBMBF, 03IS2071D, Light2Hydroge

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K+ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K+, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current

Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current

One major determinant of the efficacy of antibiotics on Gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg2+ reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg2+-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg2+. Analysis of the resulting amplitude histograms with beta distributions revealed the rate constants of blocking (k(OB)) and unblocking (k(BO)) in the range of 1,000 to 120,000 s(-1). As expected for a bimolecular reaction, k(OB) was proportional to blocker concentration and k(BO) independent of it. k(OB) was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being similar to 25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg2+ and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg2+. The difference in the accessibility of the binding sites also explains the dependency of k(OB) on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding

Phospholamban generates cation selective ion channels.

Phosholamban (PLN) is involved in the contractility of cardiac muscles by regulating the intracellular calcium concentration (Ca(2+)(cyt)) of cardiac myocytes. This occurs via a modulation of the sarco-/endoplasmic CaATPase (SERCA). In spite of high-resolution structures the molecular mode of PLN action is yet not known. In the present paper we readdress the question whether PLN proteins can generate ion channel activity. Reconstitution of PLN in planar lipid bilayers reveals single channel fluctuations, which are characterized by two conductance levels, long open/closed dwell times, moderate selectivity between monovalent cations and no perceivable Ca(2+) permeability. The PLN generated channel activity could be inhibited by a PLN antibody (abPLN) implying that the channel activity is indeed due to the inherent channel function of the PLN protein

Ca2+ block and flickering both contribute to the negative slope of the IV curve in BK channels

Single-channel current–voltage (IV) curves of human large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels are quite linear in 150 mM KCl. In the presence of Ca(2+) and/or Mg(2+), they show a negative slope conductance at high positive potentials. This is generally explained by a Ca(2+)/Mg(2+) block as by Geng et al. (2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210955) in this issue. Here, we basically support this finding but add a refinement: the analysis of the open-channel noise by means of β distributions reveals what would be found if measurements were done with an amplifier of sufficient temporal resolution (10 MHz), namely that the block by 2.5 mM Ca(2+) and 2.5 mM Mg(2+) per se would only cause a saturating curve up to +160 mV. Further bending down requires the involvement of a second process related to flickering in the microsecond range. This flickering is hardly affected by the presence or absence of Ca(2+)/Mg(2+). In contrast to the experiments reported here, previous experiments in BK channels (Schroeder and Hansen. 2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) showed saturating IV curves already in the absence of Ca(2+)/Mg(2+). The reason for this discrepancy could not be identified so far. However, the flickering component was very similar in the old and new experiments, regardless of the occurrence of noncanonical IV curves