Viral potassium channels as a robust model system for studies of membrane-protein interaction

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• 作者：Christian J. Braun^a ; Christine Lachnit^a ; Patrick Becker^a ; Leonhard M. Henkes^b ; Cristina Arrigoni^c ; Stefan M. Kast^b ; Anna Moroni^c ; ^d ; Gerhard Thiel^a ; Indra Schroeder^a ; ^{schroeder@bio.tu-darmstadt.de" class="auth_mail}
• 关键词：BLM ; black lipid membrane ; DPhPC ; 1 ; 2-diphytanoyl-sn-glycero-3-phosphocholine ; GUV ; giant unilamellar vesicle
• 刊名：Biochimica et Biophysica Acta (BBA)/Biomembranes
• 出版年：April, 2014
• 年：2014
• 卷：1838
• 期：4
• 页码：1096-1103
• 全文大小：1132 K

文摘

The viral channel Kcv_NTS belongs to the smallest K⁺ channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of Kcv_NTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80 pS), high open-probability (> 50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins鈥擟hannels for Cellular Networking.