Relevance of Lysine Snorkeling in the Outer Transmembrane Domain of Small Viral Potassium Ion Channels

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called “snorkeling” of a cationic amino acid, which is conserved in the outer TMD of small viral K+ channels. Experimentally, snorkeling activity is not mandatory for KcvPBCV-1 because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, KcvATCV-1 and KcvMT325, lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of KcvPBCV-1 and N-terminally truncated mutants; the truncated mutants mimic KcvATCV-1 and KcvMT325. Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K+ channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains

Relevance of Lysine Snorkeling in the Outer Transmembrane Domain of Small Viral Potassium Ion Channels

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called “snorkeling” of a cationic amino acid, which is conserved in the outer TMD of small viral K+ channels. Experimentally, snorkeling activity is not mandatory for KcvPBCV-1 because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, KcvATCV-1 and KcvMT325, lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of KcvPBCV-1 and N-terminally truncated mutants; the truncated mutants mimic KcvATCV-1 and KcvMT325. Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K+ channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains

Phycodnavirus Potassium Ion Channel Proteins Question the Virus Molecular Piracy Hypothesis

Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K+ channels. To determine if these viral K+ channels are the product of molecular piracy from their hosts, we compared the sequences of the K+ channel pore modules from seven phycodnaviruses to the K+ channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K+ channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K+ channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K+ channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K+ channels in algae and perhaps even all cellular organisms

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

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

Effects of ketamine, s-ketamine, and MK 801 on proliferation, apoptosis, and necrosis in pancreatic cancer cells

Background Adenocarcinoma of the pancreas is one of the most aggressive cancer diseases affecting the human body. The oncogenic potential of this type of cancer is mainly characterized by its extreme growth rate triggered by the activation of signaling cascades. Modern oncological treatment strategies aim at efficiently modulating specific signaling and transcriptional pathways. Recently, anti-tumoral potential has been proven for several substances that are not primarily used in cancer treatment. In some tumor entities, for example, administration of glutamate antagonists inhibits cell proliferation, cell cycle arrest, and finally cell death. To attain endogenic proof of NMDA receptor type expression in the pancreatic cancer cell lines PaTu8988t and Panc-1 and to investigate the impact of ketamine, s-ketamine, and the NMDA receptor antagonist MK 801 on proliferation, apoptosis, and necrosis in pancreatic carcinoma. Methods Cell proliferation was measured by means of the ELISA BrdU assay, and the apoptosis rate was analyzed by annexin V staining. Immunoblotting were also used. Results The NMDA receptor type R2a was expressed in both pancreatic carcinoma cell lines. Furthermore, ketamine, s-ketamine, and MK 801 significantly inhibited proliferation and apoptosis. Conclusions In this study, we showed the expression of the NMDA receptor type R2a in pancreatic cancer cells. The NMDA antagonists ketamine, s-ketamine, and MK 801 inhibited cell proliferation and cell death. Further clinical studies are warranted to identify the impact of these agents on the treatment of cancer patients

Stress in ballet: looking for the mind body unit for a chance to cure

Stress in ballet: looking for the mind body unit for a chance to cure OBJECTIVES The aim of this study was to analyze psychological stress, their causes and mechanisms in a population of professional dancers of a prestigious Italian company. These data may provide the basis to work out individual-centered stress prevention concepts. BACKGROUND AND AIMS Dancers usually work under major physical workload and psychological stress conditions. The aim of this study was to analyze stress risk factors, their causes and mechanisms in a population of professional dancers, and elaborate the adoption of preventive measures. MATERIALS AND METHODS The study was carried out on a sample of 38 Italian ballet dancers, from a company of a large Italian city, 26 women and 12 men aged between 18 and 56 years. We used the HSE (health and safety executive) Indicator Tool. Than we focused on the patterns of illness afflicting performing artists and explained possible management strategies. RESULTS In none of the seven organizational dimensions analyzed (demand, control, managers’ support, peer support, relationships, role and change) the required standards were achieved. In particular, the situation among female dancers is worrisome and suggests that living conditions of these patients need to be understood. CONCLUSIONS In ballet higher attention is paid to the physical resources which doesn't correspond to the psychic dimension. Hence there is requirement to develop a cultural and therapeutic approach that proposes an idea of ​​original mind-body unity like the one present in the Human Birth Theory by Massimo Fagioli

Phycodnavirus Potassium Ion Channel Proteins Question the Virus Molecular Piracy Hypothesis

Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K+ channels. To determine if these viral K+ channels are the product of molecular piracy from their hosts, we compared the sequences of the K+ channel pore modules from seven phycodnaviruses to the K+ channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K+ channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K+ channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K+ channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K+ channels in algae and perhaps even all cellular organisms

Salt bridges in the miniature viral channel Kcv are important for function.

The viral potassium channel Kcv comprises only 94 amino acids, which represent the pore module of more complex K(+) channels. As for Kir-type channels, Kcv also has a short N-terminal helix exposed to the cytoplasm, upstream of the first transmembrane domain. Here we show that this helix is relevant for Kcv function. The presence of charged amino acids, which form dynamic inter- and intra-subunit salt bridges is crucial. Electrophysiological measurements, yeast rescue experiments and molecular dynamics simulations show that mutants in which the critical salt bridge formation is impaired have no or reduced channel activity. We conclude that these salt bridges destabilise the complexation of K(+) ions by negative charges on the inner transmembrane domain at the entrance into the cavity. This feature facilitates a continuous and coordinated transfer of ions between the cavity and the cytoplasm for channels without the canonical bundle crossing

Relevance of Lysine Snorkeling in the Outer Transmembrane Domain of Small Viral Potassium Ion Channels.

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called "snorkeling" of a cationic amino acid, which is conserved in the outer TMD of small viral K(+) channels. Experimentally, snorkeling activity is not mandatory for Kcv(PBCV-1) because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, Kcv(ATCV-1) and Kcv(MT325), lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of Kcv(PBCV-1) and N-terminally truncated mutants; the truncated mutants mimic Kcv(ATCV-1) and Kcv(MT325). Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K(+) channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains