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.

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