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

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.