Structure/Function Analysis of the Viral Potassium Channel Kcv - Mutagenesis studies of the two transmembrane domains

• Main Author: Gebhardt, Manuela
• Format: Others
• Language: English; en
• Published: 2011
• Online Access: https://tuprints.ulb.tu-darmstadt.de/2391/2/Manuela_Gebhardt_2011.pdf; Gebhardt, Manuela <http://tuprints.ulb.tu-darmstadt.de/view/person/Gebhardt=3AManuela=3A=3A.html> (2011): Structure/Function Analysis of the Viral Potassium Channel Kcv - Mutagenesis studies of the two transmembrane domains.Darmstadt, Technische Universität, [Ph.D. Thesis]

The viral potassium channel Kcv from Paramecium bursaria chlorella virus 1 (PBCV-1) is with only 94 amino acids minimal in size. Indeed, Kcv is one of the smallest potassium channels known so far, but still exhibits almost structural and functional hallmarks of complex potassium ion channels. Here we analyse the importance of the two transmembrane domains (TMD) for channel function. Using an alanine-scanning approach in combination with yeast complementation and electrophysiological recordings, we identified crucial important sites in both TMDs, which are important for channel function. Many of the key amino acids are located in the outer transmembrane domain and are essential for the correct positioning of the protein in the lipid membrane. Snorkeling effects in KcvPBCV-1 are nonessential for a proper channel function: A lysine near the water/lipid interface in a TM segment is able to snorkel. This snorkeling can increase the hydrophobic length of TM segments and helps to span the lipid membrane. Computational studies of KcvPBCV-1 have shown that the lysine at position 29 in the TMD1 has to be deprotonated for proper channel function. Extensive mutational studies of the lysine at position 29 in KcvPBCV-1 have shown that all amino acid exchanges, with exception of proline, are allowed at this position. This means that KcvPBCV-1 indeed tolerates a neutral amino acid in this position without loosing function. However, when the equivalent lysine, which is highly conserved in viral channels, is substituted by alanine in the related channels KcvMT325 or KcvATCV-1, these channels loose their function. The latter two channels do not have the cytosolic N-terminal domain, which is essential in KcvPBCV-1. We therefore propose that the snorkeling effect is becoming essential in the structural context of the Kcv channels without cytosolic N-terminus, and that this feature is not crucial for the functionality of KcvPBCV-1. Aromatic amino acids in the TMD1 are crucial for the anchoring of the protein in the lipid membrane: TMD1 contains several aromatic amino acids. According to the structural model of Kcv, these aromatic side chains are facing towards the lipid membrane and anchor the channel in the membrane. The anchoring is also reflected in the distribution of the b-factors of Kcv, which are a measure for the flexibility or rigidity of the amino acids. The N-terminus of Kcv and the first half of the TMD1 exhibit high b-factors and are flexible; the rest of the TMD1, starting from His17, is rigid with low b-factors. The alanine exchange experiments underscore the functional importance of this anchoring. An exchange of the aromatic residues in TMD1 beginning with His17 greatly reduces or abolishes channel function. These negative effects on channel function can be explained by a decreased anchoring of the protein in the membrane. The π-stack between the two TMDs stabilises the spatial structure of the channel: Alanine-scanning mutagenesis together with information on the three dimensional structure of Kcv identified intramolecular interactions between the TMD1 (Phe30) and the TMD2 (His83). A π-π-interaction between aromatic rings in TMD1 and TMD2 generates a tight connection (π-stack) between the two TMDs and coordinates them into the correct position. A mutation of one of the π-stack-partners leads nearly in all cases to the loss of the channel function. Only substitutions in one partner amino acid (Phe30), which also allow π-stacking interactions (Try, Met), are still able to maintain channel function. The results of these experiments imply that the intramolecular contact between the TMDs is essential for function. The position of the π-stack in the channel model suggests, that the rigid part of TMD1 allows the stabilising of the upper part of TMD2 via this connection. The C-terminal amino acid influences the potassium concentration in the cavity: Mutations of the last C-terminal amino acid of the TMD2 in KcvPBCV-1 affect the activity of the channel. Computational data of the potassium concentration profiles of the different mutants predict that these mutations influence the internal potassium concentration of the channel. These changes do not occur, as expected, directly at the mouth of the channel but in the cavity. A theoretically predicted depletion or accumulation of potassium in the cavity, as a result of a mutation of the terminal amino acid, generates channels, which show in the experiments either a lower or no activity. Therefore, small changes in the amino acid sequence could cause drastic effects in the global K+ concentration distribution in the channel and, therewith, influences channel function. The good agreement between theory and experiment suggests that an optimal K+ concentration is essential for a proper channel function; a too high or too low K+ concentration leads to reduced or no channel function. Furthermore, the results reveal the quality of the homology model of Kcv, which enables us to find long-term interactions between the C-terminus and the cavity, an interaction, which is independent on the salt bridges at the cytosolic entrance of the channel.