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Quaternary ammonium cations are a useful tool to investigate viral potassium channels

Winterstein, Tobias (2020):
Quaternary ammonium cations are a useful tool to investigate viral potassium channels.
Darmstadt, Technische Universität, DOI: 10.25534/tuprints-00011324,
[Online-Edition: https://tuprints.ulb.tu-darmstadt.de/11324],
[Ph.D. Thesis]

Abstract

Potassium channels enable the selective and passive transport of potassium ions through membranes. They are involved in a variety of cellular and physiological processes. These include the contraction of muscle cells or the generation and transmission of action potentials. It is therefore of interest to elucidate the structure and function of these proteins. In this work, viral potassium channels of the ATCV-1 family are used for an analysis of structure function correlates. The monomers of these channels are with 82 amino extremely small but represent in spite of their miniature size still the most important structural and functional features of the pore module of complex potassium channels.

In the first part of this thesis structural differences of the two channels KcvNTS and KcvS are investigated with the help of quaternary ammonium cations (QA). Despite a sequence identity of 90%, these two channels show electrophysiological differences. These differences are due to an inner gate in KcvS, which produces long-lived closed phases and thus significantly reduces the open probability. This gate is mediated by an intrahelical hydrogen bridge of Ser77. Intracellularly added QAs cause in planar lipid bilayer recordings of both channels a voltage-dependent block, which becomes slower with increasing size of the blocker. The analysis of single channel gating in absence and presence of different QA blockers shows that the affinity of KcvS is twice as high as that of KcvNTS. It can be shown that this difference is due to the inner gate, which is apparently able to trap the QAs in the cavity through the aromatic side chain. Due to the minor differences in the association rate constants between different blockers of the two channels, no conclusions can be drawn about the pore diameter.

Goal of the second part was the engineering of a light switchable viral K+ channel by modular design. The molecule MAL-AZO-QA was bound to the KcvNTS channel by means of a maleimide bond. As a result, the charged head group should be able to block the channel in a light dependent manner. In a first step sensitivity of the channel towards Tetraethylammonium (TEA) was increased by specific mutations just outside of the selectivity filter. The consequent reduction of unitary conductance was in the next step significantly increased by a further mutation just below the filter region in the area of the cavity. In the next step the amino acid cysteine was introduced into the protein at various positions within the extracellular loop. This should ensure coupling of the light-switchable molecule to the channel at an appropriate distance to the pore. While all these preparatory steps were successful it was not possible to find the expected light sensitive blocking of the channel.

The third part of the project was dedicated to a methodological improvement of the lipid bilayer technique. In this context we examined the advantages and disadvantages of photolithographic generated pores in the epoxide ADEX as septum for lipid bilayer experiments. The functional properties of these pores for channels recording were compared to conventional septa generated in Teflon foils. The data show that the functional properties of the KcvNTS channels as a test system are identical in both septa. While the photolithographic technique allowed the generation of apertures with diameters as small as 30 µm they provided no advantages in terms of capacity or signal-to-noise ratio over larger apertures with 100 µm in Teflon. However, the advantages of the ADEX films are that they can be cleaned with acetone for frequent reuse. Even more beneficial is the observation that they are more suitable for long-term measurements since the stability of the bilayer is not compromised by pipetting of the measuring solution.

Item Type: Ph.D. Thesis
Erschienen: 2020
Creators: Winterstein, Tobias
Title: Quaternary ammonium cations are a useful tool to investigate viral potassium channels
Language: English
Abstract:

Potassium channels enable the selective and passive transport of potassium ions through membranes. They are involved in a variety of cellular and physiological processes. These include the contraction of muscle cells or the generation and transmission of action potentials. It is therefore of interest to elucidate the structure and function of these proteins. In this work, viral potassium channels of the ATCV-1 family are used for an analysis of structure function correlates. The monomers of these channels are with 82 amino extremely small but represent in spite of their miniature size still the most important structural and functional features of the pore module of complex potassium channels.

In the first part of this thesis structural differences of the two channels KcvNTS and KcvS are investigated with the help of quaternary ammonium cations (QA). Despite a sequence identity of 90%, these two channels show electrophysiological differences. These differences are due to an inner gate in KcvS, which produces long-lived closed phases and thus significantly reduces the open probability. This gate is mediated by an intrahelical hydrogen bridge of Ser77. Intracellularly added QAs cause in planar lipid bilayer recordings of both channels a voltage-dependent block, which becomes slower with increasing size of the blocker. The analysis of single channel gating in absence and presence of different QA blockers shows that the affinity of KcvS is twice as high as that of KcvNTS. It can be shown that this difference is due to the inner gate, which is apparently able to trap the QAs in the cavity through the aromatic side chain. Due to the minor differences in the association rate constants between different blockers of the two channels, no conclusions can be drawn about the pore diameter.

Goal of the second part was the engineering of a light switchable viral K+ channel by modular design. The molecule MAL-AZO-QA was bound to the KcvNTS channel by means of a maleimide bond. As a result, the charged head group should be able to block the channel in a light dependent manner. In a first step sensitivity of the channel towards Tetraethylammonium (TEA) was increased by specific mutations just outside of the selectivity filter. The consequent reduction of unitary conductance was in the next step significantly increased by a further mutation just below the filter region in the area of the cavity. In the next step the amino acid cysteine was introduced into the protein at various positions within the extracellular loop. This should ensure coupling of the light-switchable molecule to the channel at an appropriate distance to the pore. While all these preparatory steps were successful it was not possible to find the expected light sensitive blocking of the channel.

The third part of the project was dedicated to a methodological improvement of the lipid bilayer technique. In this context we examined the advantages and disadvantages of photolithographic generated pores in the epoxide ADEX as septum for lipid bilayer experiments. The functional properties of these pores for channels recording were compared to conventional septa generated in Teflon foils. The data show that the functional properties of the KcvNTS channels as a test system are identical in both septa. While the photolithographic technique allowed the generation of apertures with diameters as small as 30 µm they provided no advantages in terms of capacity or signal-to-noise ratio over larger apertures with 100 µm in Teflon. However, the advantages of the ADEX films are that they can be cleaned with acetone for frequent reuse. Even more beneficial is the observation that they are more suitable for long-term measurements since the stability of the bilayer is not compromised by pipetting of the measuring solution.

Place of Publication: Darmstadt
Divisions: 10 Department of Biology
10 Department of Biology > Plant Membrane Biophysics
Date Deposited: 19 Jan 2020 20:55
DOI: 10.25534/tuprints-00011324
Official URL: https://tuprints.ulb.tu-darmstadt.de/11324
URN: urn:nbn:de:tuda-tuprints-113240
Referees: Thiel, Prof. Dr. Gerhard and Bertl, Prof. Dr. Adam
Refereed / Verteidigung / mdl. Prüfung: 9 December 2019
Alternative Abstract:
Alternative abstract Language
Kaliumkanäle ermöglichen den selektiven und passiven Transport von Kaliumionen über Membranen. Sie sind an einer Vielzahl von zellulären und physiologischen Prozessen beteiligt. Dazu gehören die Kontraktion von Muskelzellen oder die Erzeugung und Übertragung von Aktionspotenzialen. Es ist daher von Interesse, die Struktur und Funktion dieser Proteine aufzuklären. In dieser Arbeit werden virale Kaliumkanäle der ATCV-1-Familie für eine Analyse von Struktur und Funktion verwendet. Die Monomere dieser Kanäle sind mit 82 Aminosäuren extrem klein, stellen aber trotz ihrer geringen Größe immer noch die wichtigsten strukturellen und funktionellen Merkmale des Porenmoduls komplexer Kaliumkanäle dar. Im ersten Teil dieser Arbeit werden strukturelle Unterschiede der beiden Kanäle KcvNTS und KcvS mit Hilfe von quaternären Ammoniumkationen (QAs) untersucht. Trotz einer Sequenzidentität von 90% zeigen diese beiden Kanäle elektrophysiologische Unterschiede. Diese Unterschiede sind auf ein inneres Gate in KcvS zurückzuführen, das langlebige geschlossene Phasen erzeugt und damit die Offenwahrscheinlichkeit deutlich reduziert. Dieses Gate wird durch eine intrahelikale Wasserstoffbrücke von Ser77 vermittelt. Intrazellulär zugegebene QAs bewirken im planaren Lipid-Bilayer bei beiden Kanälen einen spannungsabhängigen Block, der mit zunehmender Größe des Blockers langsamer wird. Die Analyse des Kanalschaltens in Abwesenheit und Anwesenheit verschiedener QA-Blocker zeigt, dass die Affinität von KcvS doppelt so hoch ist wie die von KcvNTS. Es kann gezeigt werden, dass dieser Unterschied auf das innere Gate zurückzuführen ist, das anscheinend in der Lage ist, die QAs durch die aromatische Seitenkette in der Kavität einzufangen. Aufgrund der geringen Unterschiede in den Assoziationsratenkonstanten zwischen den verschiedenen Blockern der beiden Kanäle können keine Rückschlüsse auf den Porendurchmesser gezogen werden. Ziel des zweiten Teils war die Entwicklung eines lichtschaltbaren viralen Kaliumkanals in Modulbauweise. Das Molekül MAL-AZO-QA wurde über eine Maleimidbindung an KcvNTS gebunden. Daher sollte die geladene Kopfgruppe in der Lage sein den Kanal lichtabhängig zu blockieren. In einem ersten Schritt wurde die Empfindlichkeit des Kanals gegenüber Tetraethylammonium (TEA) durch spezifische Mutationen außerhalb des Selektivitätsfilters erhöht. Die damit verbundene Reduktion der Leitfähigkeit wurde im nächsten Schritt durch eine weitere Mutation unmittelbar unterhalb der Filterregion im Bereich der Kavität signifikant erhöht. Im nächsten Schritt wurde die Aminosäure Cystein an verschiedenen Stellen innerhalb des extrazellulären Loops in das Protein eingebracht. Dadurch sollte die Kopplung des lichtschaltbaren Moleküls an den Kanal in einem angemessenen Abstand zur Pore gewährleistet sein. Obwohl alle diese Vorbereitungsschritte erfolgreich waren, war es nicht möglich, die erwartete lichtempfindliche Blockierung des Kanals zu erreichen. Der dritte Teil des Projekts widmete sich einer methodischen Verbesserung der Lipid-Bilayer-Technik. In diesem Zusammenhang wurden die Vor- und Nachteile photolithographisch erzeugter Poren im Epoxid ADEX als Septum für Lipid-Bilayer untersucht. Die funktionellen Eigenschaften dieser Poren wurden mit konventionellen Septen aus Teflonfolie verglichen. Die Daten zeigen, dass die funktionellen Eigenschaften von KcvNTS als Testsystem in beiden Septen identisch sind. Während die photolithographische Technik die Erzeugung von Aperturen mit Durchmessern von nur 30 µm erlaubte, boten sie gegenüber größeren Aperturen mit 100 µm in Teflon keine Vorteile in Bezug auf Kapazität oder Signal-Rausch-Verhältnis. Die Vorteile der ADEX-Folien liegen jedoch darin, dass sie bei häufiger Wiederverwendung mit Aceton gereinigt werden können. Noch vorteilhafter ist die Beobachtung, dass sie für Langzeitmessungen besser geeignet sind, da die Stabilität der Membran durch das Pipettieren der Messlösung nicht beeinträchtigt wird.German
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