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Structure/function correlates and protein/lipid interaction of the viral potassium channel KcvNTS

Braun, Christian Julian (2014)
Structure/function correlates and protein/lipid interaction of the viral potassium channel KcvNTS.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

Ion channels are present in every domain of life. They catalyze the rapid and selective flux of ions across membranes. It is well established that mutations or dysfunctions of ion channels often cause severe diseases. To understand the molecular mechanisms behind these so-called channelopathies it is necessary to understand the structure/function correlates and protein/lipid interaction of channels at the single-protein level. Planar lipid bilayer techniques, the oldest and most reduced systems for a functional characterization of ion channels, are well suited to examine basic structure/function relations in a defined lipid environment. Here we improved the performance of the conventional planar lipid bilayer technique. An air-bubble functions as a tool for the rapid creation and stabilization of bilayers and even more important for reducing the number of active channels in the bilayer for real single-channel recordings. A further technical improvement is the establishment of an in vitro (cell-free) expression system for ion channels, which supports a rapid protein production and a contamination free reconstitution. With these systems we performed a detailed single-channel analysis of the viral K+ channel KcvNTS, one of the smallest potassium channels known so far. The data show that the protein has a very high selectivity for K+ over Na+; it is permeable for Rb+ although the unitary conductance is lower than that of K+. When Cs+ is the only cation present in the buffer the channel conducts it, albeit with a low conductance. If Cs+ is present together with K+ even at a low concentration it will block the K+ inward current in a side specific and voltage dependent manner. The Cs+ block increases in strength over several minutes suggesting a slow conformational change in the protein. A further characteristic feature of the KcvNTS is a distinct pH dependency of the open probability. The latter decreases from a value close to 90% with acidification of the buffer down to ca. 10%. This pH dependent open probability is correlated with an increase in the voltage dependency of the channel suggesting a titratable amino acid in the protein, which acquires the function of a voltage sensor and presumably of a gate. Because of its small size with only 82 aa per monomer the KcvNTS protein is quasi fully embedded in the membrane. A functional test in planar lipid bilayers of different thickness, which are made from lipids with different acyl chain length (C14 - C16/18) or by adding solvents or cholesterol, shows that the channel is functional under all conditions. While the unitary conductance is not affected by the thickness the open probability is sensitive to it. With increasing bilayer thickness the channel exhibits more frequently a second gating mode, which is characterized by a voltage dependency. In the latter mode positive voltages cause a decrease in the channel open probability. The question of the molecular mechanism, which is responsible for this unusual gating in a channel without a notable charge in the electrical field, remains unanswered. Also the question why the membrane thickness affects this gating mode remains unsolved. The data however show for the first time that the lipid environment can have a dramatic effect on the voltage dependency of a protein.

Since the bilayer technique bears the hazard of artifacts from impure protein isolations and from lipid pores KcvNTS is as a control also produced and purified from Pichia pastoris and expressed heterologously in HEK293 cells. Comparing the aforementioned data of the in vitro expressed protein reconstituted in conventional planar lipid bilayers with those recorded with other methods and in particular with those measured by the conventional patch clamp technique in HEK293 cells show no large difference between the different systems. The results of these experiments stress that the obtained data with the in vitro expressed KcvNTS channel in conventional bilayers is indeed representative for the function of this channel protein. Collectively the present results show that a protein as small as the KcvNTS is able to function in a robust manner as a selective K+ channel in different membrane environments. The protein, which is equivalent to the pore module of more complex K+ channels, has inherent gating properties, which are sensitive to the pH, Cs+ and the membrane thickness. This underscores the view of multiple gates in the pore module of K+ channels.

Typ des Eintrags: Dissertation
Erschienen: 2014
Autor(en): Braun, Christian Julian
Art des Eintrags: Erstveröffentlichung
Titel: Structure/function correlates and protein/lipid interaction of the viral potassium channel KcvNTS
Sprache: Englisch
Referenten: Thiel, Professor Gerhard ; Bertl, Professor Adam
Publikationsjahr: 25 April 2014
Ort: Darmstadt, Germany
Datum der mündlichen Prüfung: 27 Juni 2014
URL / URN: http://tuprints.ulb.tu-darmstadt.de/4076
Kurzbeschreibung (Abstract):

Ion channels are present in every domain of life. They catalyze the rapid and selective flux of ions across membranes. It is well established that mutations or dysfunctions of ion channels often cause severe diseases. To understand the molecular mechanisms behind these so-called channelopathies it is necessary to understand the structure/function correlates and protein/lipid interaction of channels at the single-protein level. Planar lipid bilayer techniques, the oldest and most reduced systems for a functional characterization of ion channels, are well suited to examine basic structure/function relations in a defined lipid environment. Here we improved the performance of the conventional planar lipid bilayer technique. An air-bubble functions as a tool for the rapid creation and stabilization of bilayers and even more important for reducing the number of active channels in the bilayer for real single-channel recordings. A further technical improvement is the establishment of an in vitro (cell-free) expression system for ion channels, which supports a rapid protein production and a contamination free reconstitution. With these systems we performed a detailed single-channel analysis of the viral K+ channel KcvNTS, one of the smallest potassium channels known so far. The data show that the protein has a very high selectivity for K+ over Na+; it is permeable for Rb+ although the unitary conductance is lower than that of K+. When Cs+ is the only cation present in the buffer the channel conducts it, albeit with a low conductance. If Cs+ is present together with K+ even at a low concentration it will block the K+ inward current in a side specific and voltage dependent manner. The Cs+ block increases in strength over several minutes suggesting a slow conformational change in the protein. A further characteristic feature of the KcvNTS is a distinct pH dependency of the open probability. The latter decreases from a value close to 90% with acidification of the buffer down to ca. 10%. This pH dependent open probability is correlated with an increase in the voltage dependency of the channel suggesting a titratable amino acid in the protein, which acquires the function of a voltage sensor and presumably of a gate. Because of its small size with only 82 aa per monomer the KcvNTS protein is quasi fully embedded in the membrane. A functional test in planar lipid bilayers of different thickness, which are made from lipids with different acyl chain length (C14 - C16/18) or by adding solvents or cholesterol, shows that the channel is functional under all conditions. While the unitary conductance is not affected by the thickness the open probability is sensitive to it. With increasing bilayer thickness the channel exhibits more frequently a second gating mode, which is characterized by a voltage dependency. In the latter mode positive voltages cause a decrease in the channel open probability. The question of the molecular mechanism, which is responsible for this unusual gating in a channel without a notable charge in the electrical field, remains unanswered. Also the question why the membrane thickness affects this gating mode remains unsolved. The data however show for the first time that the lipid environment can have a dramatic effect on the voltage dependency of a protein.

Since the bilayer technique bears the hazard of artifacts from impure protein isolations and from lipid pores KcvNTS is as a control also produced and purified from Pichia pastoris and expressed heterologously in HEK293 cells. Comparing the aforementioned data of the in vitro expressed protein reconstituted in conventional planar lipid bilayers with those recorded with other methods and in particular with those measured by the conventional patch clamp technique in HEK293 cells show no large difference between the different systems. The results of these experiments stress that the obtained data with the in vitro expressed KcvNTS channel in conventional bilayers is indeed representative for the function of this channel protein. Collectively the present results show that a protein as small as the KcvNTS is able to function in a robust manner as a selective K+ channel in different membrane environments. The protein, which is equivalent to the pore module of more complex K+ channels, has inherent gating properties, which are sensitive to the pH, Cs+ and the membrane thickness. This underscores the view of multiple gates in the pore module of K+ channels.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

Ionenkanäle, welche einen schnellen und selektiven Ionenfluss über Membranen katalysieren, werden in allen Domänen des Lebens gefunden. Mutationen oder Fehlfunktionen dieser Kanäle können oft schwerwiegende Folgen haben. Um die molekularen Mechanismen hinter diesen sogenannten „Channelopathien“ zu verstehen, sind Struktur-Funktionsbeziehungen sowie Protein-Lipidinteraktionen auf Einzelkanalebene erforderlich. Zu den ältesten und reduziertesten elektrophysiologischen Messsystemen, um Proteine auf Einzelkanalniveau in einer definierten Lipidumgebung zu untersuchen, gehören die konventionellen planaren lipid bilayer Syteme. In dieser Studie verbesserten wir die konventionelle bilayer Technik mit Hilfe einer Luftblase, welche als ein Werkzeug dient, mit der ein lipid bilayer schnell aufgespannt und gleichzeitig ein unstabiler lipid bilayer stabilisiert werden kann. Des Weiteren kann mit der Luftblase die Anzahl der Ionenkanäle innerhalb der Membran aktiv reduziert werden, sodass echte Einzelkanalmessungen möglich sind. Eine weitere technische Verbesserung ist die Etablierung eines in vitro (künstliches/zellfreies) Expressionssystems, mit dem Proteine zeitsparend exprimiert und kontaminationsfrei in den lipid bilayer rekonstituiert werden können. In dieser Arbeit wird, mit den obengenannten Verbesserungen, der virale Kaliumkanal KcvNTS, einer der kleinsten bekannten Kaliumkanäle, auf Einzelkanalebene untersucht. Das Kanalprotein zeigt eine hohe Selektivität für K+ gegenüber Na+. Trotz einer geringeren Leitfähigkeit ist der Kanal auch für Rb+ permeabel. Ist Cs+ das einzige verfügbare Kation wird dieses ebenfalls geleitet. In Experimenten aus einer Mischung mit K+ und Cs+ wirkt bereits eine geringe Konzentration an Cs+ einseitig blockierend auf den Kanal; der Einwärtsstrom wird spannungsabhängig blockiert. Die Stärke des Blocks und damit die Sensitivität gegenüber Cs+ ist zeitabhängig was auf eine langsame Konformationsänderung innerhalb des Proteins zurück zu führen ist. Ein weiteres Charakteristikum des KcvNTS ist eine pH abhängige Offenwahrscheinlichkeit, welche sich in einer Verringerung von ca. 90% zu etwa 10% mit zunehmender Ansäuerung des Milieus widerspiegelt. Die pH abhängige Offenwahrscheinlichkeit korreliert mit einer zunehmenden Spannungsabhängigkeit welche zu der Annahme führt, dass im Kanal eine titrierbare Aminosäure vorliegt, die als Spannungssensor und auch als Gate fungieren kann. Mit einer geringen Größe von nur 82 Aminosäuren pro Monomer ist KcvNTS quasi vollständig in der Lipidmembran eingebettet. Veränderungen der Membrandicke durch Variationen der Fettsäurelängen der Lipide (C14 - C16/18), durch Lösungsmittel oder durch Zugabe von Cholesterol zeigen, dass der Kanal in jeder Umgebung funktionsfähig ist. Die Leitfähigkeit des Kanalproteins wird nicht durch die Membrandicke beeinflusst, jedoch die Offenwahrscheinlichkeit. Dies zeigt sich in einem alternativen, spannungsabhängigen Gating des Kanals. Mit zunehmend positiven Spannungen nimmt die Offenwahrscheinlichkeit ab. Der molekulare Mechanismus hinter einem spannungsabhängigen Gating, in einem Kanal ohne nennenswerte Ladung im elektrischen Feld, bleibt unbeantwortet. Des Weiteren ist nicht geklärt in wieweit unterschiedliche Membrandicken Einfluss auf diesen Gating-Mechanismus haben. Dennoch zeigen diese Daten zum Ersten mal, dass die Lipidumgebung einen drastischen Effekt auf die Spannungsabhängigkeit eines Proteins hat. In Einzelkanalmessungen besteht die Gefahr von Artefakten darin, dass Proteine unsauber aufgereinigt sind oder Lipidporen die Messungen erschweren. Aus diesem Grund haben wir, neben des in vitro exprimierten KcvNTS, denselben Kanal in Pichia pastoris sowie heterolog in HEK293 Zellen exprimiert und diese Daten miteinander verglichen. Die Ergebnisse zeigen, dass das Kanalschalten des in vitro exprimierten KcvNTS in der Tat auf die Funktion des Kanals zurück zu führen ist. Zusammenfassend zeigen die Daten, dass ein kleines Protein, wie der KcvNTS, in einer robusten Weise als selektiver K+ Kanal funktionieren kann. Dieses Kanalprotein alleine, welches bei komplexeren K+ Kanäle lediglich als Porenmodul fungiert, zeigt Gating-Eigenschaften die sensitiv gegenüber pH, Cs+ und der Membrandicke sind. Diese Eigenschaften heben den Standpunkt mehrerer Gates im Porenmodul von K+ Kanälen hervor.

Deutsch
URN: urn:nbn:de:tuda-tuprints-40761
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie > Plant Membrane Biophyscis (am 20.12.23 umbenannt in Biologie der Algen und Protozoen)
10 Fachbereich Biologie
Hinterlegungsdatum: 27 Jul 2014 19:55
Letzte Änderung: 27 Jul 2014 19:55
PPN:
Referenten: Thiel, Professor Gerhard ; Bertl, Professor Adam
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 27 Juni 2014
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