Transmembrane domain length of viral K+ channels is a signal for mitochondria targeting

K+ channels operate in the plasma membrane and in membranes of organelles including mitochondria. The mechanisms and topogenic information for their differential synthesis and targeting is unknown. This article describes 2 similar viral K+ channels that are differentially sorted; one protein (Kesv) is imported by the Tom complex into the mitochondria, the other (Kcv) to the plasma membrane. By creating chimeras we discovered that mitochondrial sorting of Kesv depends on a hierarchical combination of N- and C-terminal signals. Crucial is the length of the second transmembrane domain; extending its C terminus by \u3e2 hydrophobic amino acids redirects Kesv from the mitochondrial to the plasma membrane. Activity of Kesv in the plasma membrane is detected electrically or by yeast rescue assays only after this shift in sorting. Hence only minor structural alterations in a transmembrane domain are sufficient to switch sorting of a K+ channel between the plasma membrane and mitochondria

Studien zur Funktion von Kaliumkanälen aus Viren eukaryotischer Algen (Phycodnaviridae)

Im Genom von zwei entfernt verwandten Algenviren der Familie Phycodnaviridae wurden Kaliumkanal-ähnliche Gene gefunden. Das Paramecium bursaria Chlorellavirus kodiert Kcv, den ersten bekannten viralen Kaliumkanal. Im Genom von Ectocarpus siliculosus Virus kodiert das Offene Leseraster 223 einen weiteren möglichen viralen Kaliumkanal, Kev. Durch die heterologe Expression eines chimären Proteins aus Kcv und der Pore von Kev konnte in dieser Arbeit die Funktionalität der Kev-Kanalpore gezeigt werden. Dennoch konnte durch die heterologe Expression des Kev-wildtyp-Proteins in Säugerzellen (HEK293 und CHO) oder in Oozyten von Xenopus laevis keine spezifische Kaliumkanalaktivität an der Plasmamembran dieser Zellen nachgewiesen werden. Als Ursache für die fehlende selektive Kanalaktivität kommt die intrazelluläre Verteilung des Proteins im Expressionssystem in Frage: Confokal-mikroskopische Aufnahmen von HEK293-Zellen, die Kev-GFP Fusionsprotein exprimierten, zeigen punktförmige Fluoreszenzmaxima. Im Gegensatz dazu akkumulierte die Fluoreszenz von Kcv-GFP Fusionsprotein in tubulären, membranartigen Strukturen. Durch eine Verlängerung der carboxy-terminalen Transmembranhelix von Kev-GFP-Fusionsprotein um drei oder sechs Aminosäurereste konnte jedoch dasselbe Verteilungsmuster wie mit Kcv-GFP-Fusionsprotein hergestellt werden. Die Verteilung der untersuchten viralen Kanäle wird also von der Länge der carboxy-terminalen Transmembranhelix bestimmt. Diese Ergebnisse weisen auf eine mögliche physiologische Rolle des Kev-Proteins in der dünnen Membran des Endoplasmatischen Retikulums (ER) der Wirtszelle oder in der inneren Virusmembran von Ectocarpusviren hin, welche vom ER des Wirts abgeleitet ist. In der vorliegenden Arbeit fanden sich auch Hinweise dafür, dass der Kaliumkanal Kcv aus Chlorellaviren im Viruspartikel lokalisiert ist: (i) Kcv-mRNA wird in der späten Phase der Virusreplikation synthetisiert; während dieser Phase findet auch die Assemblierung von neuen Viruspartikeln statt. (ii) Weiter konnte eine Depolarisation des Membranpotentials bei der Infektion von Chlorella NC64A durch Chlorellaviren mit dem spannungsabhängigen Fluoreszenzfarbstoff Bis-Oxonol gemessen werden. Diese Depolarisation zeigte eine sehr ähnliche Pharmakologie wie die Kanalaktivität von Kcv in heterologen Expressionssystemen und die Replikation der Viren (in plaque-Tests). Dies deutet auf eine essentielle Funktion von Kcv während der ersten Minuten der Infektion durch Chlorellaviren hin

Nicotinamide Riboside—The Current State of Research and Therapeutic Uses

Nicotinamide riboside (NR) has recently become one of the most studied nicotinamide adenine dinucleotide (NAD+) precursors, due to its numerous potential health benefits mediated via elevated NAD+ content in the body. NAD+ is an essential coenzyme that plays important roles in various metabolic pathways and increasing its overall content has been confirmed as a valuable strategy for treating a wide variety of pathophysiological conditions. Accumulating evidence on NRs’ health benefits has validated its efficiency across numerous animal and human studies for the treatment of a number of cardiovascular, neurodegenerative, and metabolic disorders. As the prevalence and morbidity of these conditions increases in modern society, the great necessity has arisen for a rapid translation of NR to therapeutic use and further establishment of its availability as a nutritional supplement. Here, we summarize currently available data on NR effects on metabolism, and several neurodegenerative and cardiovascular disorders, through to its application as a treatment for specific pathophysiological conditions. In addition, we have reviewed newly published research on the application of NR as a potential therapy against infections with several pathogens, including SARS-CoV-2. Additionally, to support rapid NR translation to therapeutics, the challenges related to its bioavailability and safety are addressed, together with the advantages of NR to other NAD+ precursors

Nicotinamide Riboside—The Current State of Research and Therapeutic Uses

Nicotinamide riboside (NR) has recently become one of the most studied nicotinamide adenine dinucleotide (NAD+) precursors, due to its numerous potential health benefits mediated via elevated NAD+ content in the body. NAD+ is an essential coenzyme that plays important roles in various metabolic pathways and increasing its overall content has been confirmed as a valuable strategy for treating a wide variety of pathophysiological conditions. Accumulating evidence on NRs’ health benefits has validated its efficiency across numerous animal and human studies for the treatment of a number of cardiovascular, neurodegenerative, and metabolic disorders. As the prevalence and morbidity of these conditions increases in modern society, the great necessity has arisen for a rapid translation of NR to therapeutic use and further establishment of its availability as a nutritional supplement. Here, we summarize currently available data on NR effects on metabolism, and several neurodegenerative and cardiovascular disorders, through to its application as a treatment for specific pathophysiological conditions. In addition, we have reviewed newly published research on the application of NR as a potential therapy against infections with several pathogens, including SARS-CoV-2. Additionally, to support rapid NR translation to therapeutics, the challenges related to its bioavailability and safety are addressed, together with the advantages of NR to other NAD+ precursors

Possible function for virus encoded K+ channel Kcv in the replication of chlorella virus PBCV-1

AbstractThe K+ channel Kcv is encoded by the chlorella virus PBCV-1. There is evidence that this channel plays an essential role in the replication of the virus, because both PBCV-1 plaque formation and Kcv channel activity in Xenopus oocytes have similar sensitivities to inhibitors. Here we report circumstantial evidence that the Kcv channel is important during virus infection. Recordings of membrane voltage in the host cells Chlorella NC64A reveal a membrane depolarization within the first few minutes of infection. This depolarization displays the same sensitivity to cations as Kcv conductance; depolarization also requires the intact membrane of the virion. Together these data are consistent with the idea that the virus carries functional K+ channels in the virion and inserts them into the host cell plasma membrane during infection

Genetic diversity in chlorella viruses flanking kcv, a gene that encodes a potassium ion channel protein

AbstractThe chlorella virus PBCV-1 encodes a 94-amino acid protein named Kcv that produces a K+-selective and slightly voltage-sensitive conductance when expressed in heterologous systems. As reported herein, (i) Northern analysis of kcv expression in PBCV-1-infected cells revealed a complicated pattern suggesting that the gene might be transcribed as a di- or tri-cistronic mRNA both at early and late times after virus infection. (ii) The protein kinase inhibitors H-89, A3, and staurosporine inhibited PBCV-1 Kcv activity in Xenopus oocytes, suggesting that Kcv activity might be controlled by phosphorylation or dephosphorylation. (iii) The PBCV-1 genomic sequence revealed a gene encoding a putative protein kinase (pkx) adjacent to kcv. These findings prompted us to examine the kcv flanking regions in 16 additional chlorella viruses and transcription in two of these viruses, as well as the effect of the three protein kinase inhibitors on two Kcv homologs in Xenopus oocytes. The results indicate (i) pkx is always located 5′ to kcv, but the spacing between the two genes varies from 31 to 1588 nucleotides. More variation occurs in the kcv 3′ flanking region of the 16 viruses. (ii) The kcv gene is expressed as a late mono-cistronic mRNA. (iii) Unlike the affect on PBCV-1 Kcv, the three protein kinase inhibitors have little or no effect on the activity of the two Kcv homologs in oocytes. (iv) A comparison of the kcv 5′ upstream sequences from the 16 viruses identified a highly conserved 10-nucleotide sequence that is present in the promoter region of all of the viruses

Potassium Ion Channels of Chlorella Viruses Cause Rapid Depolarization of Host Cells during Infection

Previous studies have established that chlorella viruses encode K(+) channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs(+) to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of Chlorella NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [t(1/2)], about 9 min) and slowest with virus NY-2A (t(1/2), about 12 min). Cs(+) inhibited membrane depolarization only in viruses that encode a Cs(+)-sensitive K(+) channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of Chlorella cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K(+) conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host