The channel activities of the full-length prion and truncated proteins

Prion disease is a fatal neurodegenerative disorder, in which the cellular prion protein PrPC is converted to a misfolded prion which in turn is hypothesized to permeabilize cellular membranes. The pathways leading to toxicity in prion disease are not yet completely elucidated and whether it also includes formation of membrane pores remains to be answered. Prion protein consists of two domains: a globular domain (GD) and a flexible N-terminus (FT) domain. Although a proximal nine polybasic amino acid (FT(23-31)) sequence of FT is a prerequisite for cellular membrane permeabilization, other functional domain regions may influence FT(23-31) and its permeabilization. By using single-channel electrical recordings, we reveal that FT(23-50) dominates the membrane permeabilization within the full-length mouse PrP (mPrP(23-230)). The other domain of FT(51-110) or C-terminal domain down-regulates the channel activity of FT(23-50) and the full-length mouse PrP (mPrP(23-230)). The addition of prion mimetic antibody, POM1 significantly enhances mPrP(23-230) membrane permeabilization, whereas POM1-Y104A, a POM1 mutant that binds to PrP but cannot elicit toxicity has negligible effect on membrane permeabilization. Additionally, anti-N-terminal antibody POM2 or Cu2+ stabilizes FT domain, thus provoking FT(23-110) channel activity. Furthermore, our setup provides a more direct method without an external fused protein to study the channel activity of truncated PrP in the lipid membranes. We therefore hypothesize that the primary N-terminal residues are essential for membranes permeabilization and other functional segments play a vital role to modulate the pathological effects of PrP-medicated neurotoxicity. This may yield essential insights into molecular mechanisms of prion neurotoxicity to cellular membranes in prion disease.

LY294002 Inhibits Intermediate Conductance Calcium-Activated Potassium (KCa3.1) Current in Human Glioblastoma Cells

Glioblastomas (GBs) are among the most common tumors with high malignancy and invasiveness of the central nervous system. Several alterations in protein kinase and ion channel activity are involved to maintain the malignancy. Among them, phosphatidylinositol 3-kinase (PI3K) activity and intermediate conductance calcium-activated potassium (KCa3.1) current are involved in several aspects of GB biology. By using the electrophysiological approach and noise analysis, we observed that KCa3.1 channel activity is LY294002-sensitive and Wortmannin-resistant in accordance with the involvement of PI3K class IIβ (PI3KC2β). This modulation was observed also during the endogenous activation of KCa3.1 current with histamine. The principal action of PI3KC2β regulation was the reduction of open probability in intracellular free calcium saturating concentration. An explanation based on the “three-gate” model of the KCa3.1 channel by PI3KC2β was proposed. Based on the roles of KCa3.1 and PI3KC2β in GB biology, a therapeutic implication was suggested to prevent chemo- and radioresistance mechanisms.

Influenza A Virus Infection Activates NLRP3 Inflammasome through Trans-Golgi Network Dispersion

The NLRP3 inflammasome consists of NLRP3, ASC, and pro-caspase-1 and is an important arm of the innate immune response against influenza A virus (IAV) infection. Upon infection, the inflammasome is activated, resulting in the production of IL-1β and IL-18, which recruits other immune cells to the site of infection. It has been suggested that in the presence of stress molecules such as nigericin, the trans-Golgi network (TGN) disperses into small puncta-like structures where NLRP3 is recruited and activated. Here, we investigated whether IAV infection could lead to TGN dispersion, whether dispersed TGN (dTGN) is responsible for NLRP3 inflammasome activation, and which viral protein is involved in this process. We showed that the IAV causes dTGN formation, which serves as one of the mechanisms of NLRP3 inflammasome activation in response to IAV infection. Furthermore, we generated a series of mutant IAVs that carry mutations in the M2 protein. We demonstrated the M2 proton channel activity, specifically His37 and Trp41 are pivotal for the dispersion of TGN, NLRP3 conformational change, and IL-1β induction. The results revealed a novel mechanism behind the activation and regulation of the NLRP3 inflammasome in IAV infection.

TACAN is a novel regulator of PKD2

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in membrane receptor PKD1 or cation channel PKD2. TACAN (also named TMEM120A), recently reported as an ion channel in neuron cells for mechano and pain sensing, is also distributed in diverse non-neuronal tissues such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes we found that TACAN inhibited the channel activity of PKD2 gain-of-function mutant F604P. The first and last transmembrane domains of TACAN were found to interact with the PKD2 C- and N-terminal portions, respectively. We showed that the TACAN N-terminus acted as a blocking peptide and that TACAN inhibits the PKD2 function through the PKD2/TACAN binding. By patch clamping in mammalian cells, we found that TACAN inhibits both the single channel conductance and open probability of PKD2 and mutant F604P. Further, PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechano sensitivity of the PKD2/TACAN channel complex.

Functional Analyses of the Two Distinctive Types of Two-Pore Channels (TPCs) and the Slow Vacuolar Channel in Marchantia polymorpha

The two-pore channel (TPC) family is widely conserved in eukaryotes. Many vascular plants, including Arabidopsis and rice, possess a single TPC gene which functions as a slow vacuolar (SV) channel—voltage-dependent cation-permeable channel located in the vacuolar membrane (tonoplast). On the other hand, a liverwort Marchantia polymorpha genome encodes three TPC homologs: MpTPC1 is similar to TPCs in vascular plants (type 1 TPC), while MpTPC2 and MpTPC3 are classified into a distinctive group (type 2 TPC). Phylogenetic analysis suggested that the type 2 TPC emerged before the land colonization in plant evolution and was lost in vascular plants and hornworts. All of the three MpTPCs were shown to be localized at the tonoplast. We generated knockout mutants of tpc1, tpc2, tpc3, and tpc2 tpc3 double mutant by CRISPR/Cas9 genome editing and performed patch-clamp analyses of isolated vacuoles. The SV channel activity was abolished in the Mptpc1 loss-of-function mutant (Mptpc1-1KO), while Mptpc2-1KO, Mptpc3-1KO, and Mptpc2-2/tpc3-2KO double mutant exhibited similar activity to the wild type, indicating that MpTPC1 (type 1) is solely responsible for the SV channel activity. Activators of mammalian TPC channels, PI(3,5)P2, and, NAADP, did not affect the ion channel activity of any MpTPCs. These results indicate that the type 1 TPCs, which are well conserved in all land plant species, encode the SV channel, while the type 2 TPCs likely encode other tonoplast cation channel(s) distinct from the SV channel and animal TPC channels.

The current threatening trends in the use of Telegram channels to the damage of state interests

The principles, conditions and features of Telegram messenger operation are revealed. The state interests in the information sphere in the conditions of spreading of social Internet services and Telegram channels are defined. The tendencies of using anonymous messengers, especially pro-Russian Telegram channels, are outlined. Ukraine’s rating of resilience to Russian misinformation has been determined. The possibilities of Telegram channels for manipulating public opinion and spreading misinformation are detailed. Threats of the functioning of anonymous Telegram channels in Ukraine have been identified. The state’s achievements in blocking banned content are highlighted. The directions of the improvement of the level of media literacy and digital awareness of the population are summarized. The necessity of legislative regulation of the Telegram channel activity in Ukraine is substantiated.

Spatiotemporal Stop-and-go Dynamics of the Mitochondrial TOM Core Complex Correlates With Three-state Channel Activity

Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. Here, we show the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, we find that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. Freely diffusing TOM-CC molecules are observed in a high permeability state, while non-moving molecules are in an intermediate and a low permeability state. We explain this behavior by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two β-barrel pores of TOM-CC. TOM-CC could thus be the first β-barrel protein channel to exhibit membrane state-dependent mechanosensitive properties.

High-Throughput Functional Analysis of CFTR and Other Apically Localized Proteins in iPSC-Derived Human Intestinal Organoids

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.