title: A library of variable viral K⁺ channel proteins from environmental sources provides insights into structure/function correlates in these proteins
Creators: name: Mach, Sebastian
Publisher: name: UNSPECIFIED
Year: 2025

description: Potassium channels are crucial transmembrane proteins that play a vital role in various physiological processes by catalyzing the passive and selective transport of potassium ions across cell membranes. These channels are universally characterized by a central, water-filled conductive pore and are found in a wide range of organisms, including eukaryotes, archaea, bacteria, and even viruses. In cellular systems, potassium channels participate in processes such as the generation and transmission of action potentials, muscle contraction, regulation of cell volume, and secretion. Understanding the molecular mechanisms underlying these functions is essential for gaining insights into both physiology and pathophysiological conditions at the cellular level. Viral potassium channels are of particular interest because they are reduced to their essential components, typically comprising fewer than 100 amino acids per monomer, representing the pore module of all complex potassium channels. Despite their small size and simplicity, they retain the key functional properties of higher evolved potassium channels, making them an ideal model for studying the structure/function relationships that regulate ion permeability and gating. This thesis focuses on deepening our understanding of these structure/function relationships. In this work, multiple newly discovered potassium (K⁺) channel-like sequences were aligned and compared to the viral reference channels Kcv_NTS (Potassium Channel from Chlorella Virus Next to Smith) and Kcv_PBCV-1 (Potassium Channel from Paramecium bursaria Chlorella Virus), and their electrophysiological properties were analyzed with respect to these reference channels. The primary experimental approach was the planar lipid bilayer (PLB) technique, which allows for a detailed electrophysiological characterization of small viral potassium channels at the level of single-channel gating events. This technique is particularly advantageous due to its cell-free nature with a well-defined lipid environment, allowing for the controlled manipulation of parameters such as ion concentration, lipid composition, and the application of K⁺ channel-specific blockers. In the first part of the study, six viral potassium channels from four different Kcv virus families, which show high sequence similarity to the reference channels Kcv_NTS and Kcv_PBCV-1, were electrophysiologically characterized and compared to the properties of these reference channels. In previous studies, point mutations had been introduced into the aforementioned reference channels, generating distinct impacts on channel function. The present work confirms that viral K⁺ channels in which these amino acid exchanges occur naturally, exhibit the same functional properties as the artificially mutated variants. For instance, the present results confirm that mutations in the pore helix stabilize the filter gate and increase the filter's sensitivity to the intracellularly applied K⁺ channel-specific blocker barium. Furthermore, another channel was found to have a naturally occurring amino acid substitution that reinstates the inner gate at a critical position at the intracellular pore entrance. Collectively, the data underscore that these functional impacts are indeed due to single point mutations and not to differences of other amino acid substitutions within the channel scaffold. The second part of the thesis reports the discovery of the first functional K2P-type channel encoded by a virus. K2P channels consist of two monomer units fused into a tandem through a linker, forming a channel pore through dimerization. Phylogenetic analyses suggest that the two pore-forming monomer units (P1 and P2) of the newly identified viral K⁺ channel K_POV, which lacks an extracellular outer cap domain, likely arose through gene duplication, indicating a viral origin for the channel. The data confirm that the viral protein, which is much smaller than any K2P channel from eukaryotes, is functional despite its small size and lack of cytosolic domains. Electrophysiological measurements further reveal that K_POV exhibits typical potassium channel properties, including gating, high potassium selectivity, and blockability by the K⁺-channel-specific blocker Ba²⁺. The third section of this work focuses on a potential gating mechanism in the viral potassium channel Kcv_NTS. Aromatic side chains of Phenylalanines are known to form hydrophobic barriers for ions in the cavities of many more complex K⁺ channels that can function as gates. In Kcv_NTS, such a Phenylalanine is located in the water-filled cavity of the protein. In analogy to the suspected function in other K⁺ channels, the aromatic amino acid in the present Kcv channel may also act as a hydrophobic barrier for passing potassium ions. However, electrophysiological experiments on Kcv_NTS mutants in which the critical Phenylalanine was replaced by Alanine revealed that this amino acid is indeed critical for function. The data, however, are not compatible with a gating function; they suggest a structural role in stabilizing the protein and preventing prolonged channel closures. Furthermore, dwell-time analysis demonstrated that removing the aromatic side chain resulted in long closed times in the Kcv_NTS F71A mutant, presumably as a result of a destabilization of the channel. This destabilization could not be reversed by the application of channel-specific blockers, which are known to stabilize the filter gate of this channel. The final section of this thesis explores another gating mechanism in a Kcv-type channel, specifically the time- and voltage-dependent inactivation process in Kcv_NH S77G (Potassium channel form Chlorella Virus New ampshire). Previous work had demonstrated that Kcv_NH S77G displays ultra-long closed states at hyperpolarizing voltages, drastically lowering its open probability and rendering it a strong outward rectifier. The positive charge of a Lysine at position 55 in the extracellular turret was suspected to drive these extended closed states. This hypothesis was based on the finding that substitution of this Lysine with the nonpolar amino acid Alanine or polar Cysteine eliminated these long closed states and their voltage dependence, while replacement with the similarly long, positively charged Arginine enhanced the inactivation process. In order to test, if a positively charged side chain at amino acid position 55 is sufficient to cause voltage-dependent long-living closures in Kcv_NH S77G at negative membrane potentials, a positive charge was here reintroduced into the voltage-independent mutant Kcv_NH S77G K55C, either by covalent binding of the molecule Mal-Azo-Qa or through complexing silver cations. Despite these modifications, the positive charge failed to re-establish the inactivation process in Kcv_NH S77G K55C. As a result, the exact mechanism behind this time- and voltage-dependent gating process remains unresolved.