P2Y2 receptor decreases blood pressure by inhibiting ENaC.

Stimulating the Gq-coupled P2Y2 receptor (P2ry2) lowers blood pressure. Global knockout of P2ry2 increases blood pressure. Vascular and renal mechanisms are believed to participate in P2ry2 effects on blood pressure. To isolate the role of the kidneys in P2ry2 effects on blood pressure and to reveal the molecular and cellular mechanisms of this action, we test here the necessity of the P2ry2 and the sufficiency of Gq-dependent signaling in renal principal cells to the regulation of the epithelial Na+ channel (ENaC), sodium excretion, and blood pressure. Activating P2ry2 in littermate controls but not principal cell-specific P2ry2-knockout mice decreased the activity of ENaC in renal tubules. Moreover, deletion of P2ry2 in principal cells abolished increases in sodium excretion in response to stimulation of P2ry2 and compromised the normal ability to excrete a sodium load. Consequently, principal cell-specific knockout of P2ry2 prevented decreases in blood pressure in response to P2ry2 stimulation in the deoxycorticosterone acetate-salt (DOCA-salt) model of hypertension. In wild-type littermate controls, such stimulation decreased blood pressure in this model of hypertension by promoting a natriuresis. Pharmacogenetic activation of Gq exclusively in principal cells using targeted expression of Gq-designer receptors exclusively activated by designer drugs and clozapine N-oxide decreased the activity of ENaC in renal tubules, promoting a natriuresis that lowered elevated blood pressure in the DOCA-salt model of hypertension. These findings demonstrate that the kidneys play a major role in decreasing blood pressure in response to P2ry2 activation and that inhibition of ENaC activity in response to P2ry2-mediated Gq signaling lowered blood pressure by increasing renal sodium excretion.

NOXA1-dependent NADPH oxidase 1 signaling mediates angiotensin II activation of the epithelial sodium channel.

The activity of the epithelial Na+ channel (ENaC) in principal cells of the distal nephron fine-tunes renal Na+ excretion. The renin-angiotensin-aldosterone system modulates ENaC activity to control blood pressure, in part, by influencing Na+ excretion. NADPH oxidase activator 1-dependent NADPH oxidase 1 (NOXA1/NOX1) signaling may play a key role in angiotensin II (ANG II)-dependent activation of ENaC. The present study aimed to explore the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC in renal principal cells. Patch-clamp electrophysiology and principal cell-specific Noxa1 knockout (PC-Noxa1 KO) mice were used to determine the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC. The activity of ENaC in the luminal plasma membrane of principal cells was quantified in freshly isolated split-opened tubules using voltage-clamp electrophysiology. ANG II significantly increased ENaC activity. This effect was robust and observed in response to both acute (40 min) and more chronic (48-72 h) ANG II treatment of isolated tubules and mice, respectively. Inhibition of ANG II type 1 receptors with losartan abolished ANG II-dependent stimulation of ENaC. Similarly, treatment with ML171, a specific inhibitor of NOX1, abolished stimulation of ENaC by ANG II. Treatment with ANG II failed to increase ENaC activity in principal cells in tubules isolated from the PC-Noxa1 KO mouse. Tubules from wild-type littermate controls, though, retained their ability to respond to ANG II with an increase in ENaC activity. These results indicate that NOXA1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells. As such, NOXA1/NOX1 signaling in the distal nephron plays a central role in Na+ homeostasis and control of blood pressure, particularly as it relates to regulation by the renin-ANG II axis.NEW & NOTEWORTHY Activity of the epithelial Na+ channel (ENaC) in the distal nephron fine-tunes renal Na+ excretion. Angiotensin II (ANG II) has been reported to enhance ENaC activity. Emerging evidence suggests that NADPH oxidase (NOX) signaling plays an important role in the stimulation of ENaC by ANG II in principal cells. The present findings indicate that NOX activator 1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells.

Chronic activation of vasopressin-2 receptors induces hypertension in Liddle mice by promoting Na+ and water retention.

The renin-angiotensin-aldosterone and arginine vasopressin-V2 receptor-aquaporin-2 (AQP2) systems converge on the epithelial Na+ channel (ENaC) to regulate blood pressure and plasma tonicity. Although it is established that V2 receptors initiate renal water reabsorption through AQP2, whether V2 receptors can also induce renal Na+ retention through ENaC and raise blood pressure remains an open question. We hypothesized that a specific increase in V2 receptor-mediated ENaC activity can lead to high blood pressure. Our approach was to test effects of chronic activation of V2 receptors in Liddle mice, a genetic mouse model of high ENaC activity, and compare differences in ENaC activity, urine Na+ excretion, and blood pressure with control mice. We found that ENaC activity was elevated in Liddle mice and could not be stimulated further by administration of desmopressin (dDAVP), a V2 receptor-specific agonist. In contrast, Liddle mice showed higher levels of expression of AQP2 and aquaporin-3, but they could still respond to dDAVP infusion by increasing phospho-AQP2 expression. With dDAVP infusion, Liddle mice excreted smaller urine volume and less urine Na+ and developed higher blood pressure compared with control mice; this hypertension was attenuated with administration of the ENaC inhibitor benzamil. We conclude that V2 receptors contribute to hypertension in the Liddle mouse model by promoting primary Na+ and concomitant water retention.NEW & NOTEWORTHY Liddle syndrome is a classic model for hypertension from high epithelial Na+ channel (ENaC) activity. In the Liddle mouse model, vasopressin-2 receptors stimulate both ENaC and aquaporin-2, which increases Na+ and water retention to such an extent that hypertension ensues. Liddle mice will preserve plasma tonicity at the expense of a higher blood pressure; these data highlight the inherent limitation in which the kidney must use ENaC as a pathway to regulate both plasma tonicity and blood pressure.

Structural and Functional Characterization of a Novel Scorpion Toxin that Inhibits NaV1.8 via Interactions With the DI Voltage Sensor and DII Pore Module.

Voltage-gated sodium channel NaV1.8 regulates transmission of pain signals to the brain. While NaV1.8 has the potential to serve as a drug target, the molecular mechanisms that shape NaV1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating NaV structure-function relationships. Arizona bark scorpions produce Na+ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit NaV1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na+ current recorded from a recombinant grasshopper mouse NaV1.8 channel (OtNaV1.8). Toxin NaTx36 hyperpolarized OtNaV1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 - S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 - S2 asparagine (N) stabilizes the NaTx36 - OtNaV1.8 complex while residues in the DI S3 - S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 - SS1 pore loop instead of the SS2 - S6 loop; the DII SS2 - S6 loop motif (QVSE) alters the conformation of the DII S5 - SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 - SS1 pore loop. Few toxins have been identified that modify NaV1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNaV1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.

Optogenetic Control of PIP2 Interactions Shaping ENaC Activity.

The activity of the epithelial Na+ Channel (ENaC) is strongly dependent on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 binds two distinct cationic clusters within the N termini of ß- and γ-ENaC subunits (ßN1 and γN2). The affinities of these sites were previously determined using short synthetic peptides, yet their role in sensitizing ENaC to changes in PIP2 levels in the cellular system is not well established. We addressed this question by comparing the effects of PIP2 depletion and recovery on ENaC channel activity and intracellular Na+ levels [Na+]i. We tested effects on ENaC activity with mutations to the PIP2 binding sites using the optogenetic system CIBN/CRY2-OCRL to selectively deplete PIP2. We monitored changes of [Na+]i by measuring the fluorescent Na+ indicator, CoroNa Green AM, and changes in channel activity by performing patch clamp electrophysiology. Whole cell patch clamp measurements showed a complete lack of response to PIP2 depletion and recovery in ENaC with mutations to ßN1 or γN2 or both sites, compared to wild type ENaC. Whereas mutant ßN1 also had no change in CoroNa Green fluorescence in response to PIP2 depletion, γN2 did have reduced [Na+]i, which was explained by having shorter CoroNa Green uptake and half-life. These results suggest that CoroNa Green measurements should be interpreted with caution. Importantly, the electrophysiology results show that the ßN1 and γN2 sites on ENaC are each necessary to permit maximal ENaC activity in the presence of PIP2.

SARS-CoV-2 infection enhances mitochondrial PTP complex activity to perturb cardiac energetics.

SARS-CoV-2 is a newly identified coronavirus that causes the respiratory disease called coronavirus disease 2019 (COVID-19). With an urgent need for therapeutics, we lack a full understanding of the molecular basis of SARS-CoV-2-induced cellular damage and disease progression. Here, we conducted transcriptomic analysis of human PBMCs, identified significant changes in mitochondrial, ion channel, and protein quality-control gene products. SARS-CoV-2 proteins selectively target cellular organelle compartments, including the endoplasmic reticulum and mitochondria. M-protein, NSP6, ORF3A, ORF9C, and ORF10 bind to mitochondrial PTP complex components cyclophilin D, SPG-7, ANT, ATP synthase, and a previously undescribed CCDC58 (coiled-coil domain containing protein 58). Knockdown of CCDC58 or mPTP blocker cyclosporin A pretreatment enhances mitochondrial Ca2+ retention capacity and bioenergetics. SARS-CoV-2 infection exacerbates cardiomyocyte autophagy and promotes cell death that was suppressed by cyclosporin A treatment. Our findings reveal that SARS-CoV-2 viral proteins suppress cardiomyocyte mitochondrial function that disrupts cardiomyocyte Ca2+ cycling and cell viability.

Renal NOXA1/NOX1 Signaling Regulates Epithelial Sodium Channel and Sodium Retention in Angiotensin II-induced Hypertension.

Aims: NADPH oxidase (NOX)-derived reactive oxygen species (ROS) are implicated in the pathophysiology of hypertension in chronic kidney disease patients. Genetic deletion of NOX activator 1 (Noxa1) subunit of NOX1 decreases ROS under pathophysiological conditions. Here, we investigated the role of NOXA1-dependent NOX1 activity in the pathogenesis of angiotensin II (Ang II)-induced hypertension (AIH) and possible involvement of abnormal renal function. Results: NOXA1 is present in epithelial cells of Henle's thick ascending limb and distal nephron. Telemetry showed lower basal systolic blood pressure (BP) in Noxa1-/-versus wild-type mice. Ang II infusion for 1 and 14 days increased NOXA1/NOX1 expression and ROS in kidney of male but not female wild-type mice. Mean BP increased 30 mmHg in wild-type males, with smaller increases in Noxa1-deficient males and wild-type or Noxa1-/- females. In response to an acute salt load, Na+ excretion was similar in wild-type and Noxa1-/- mice before and 14 days after Ang II infusion. However, Na+ excretion was delayed after 1-2 days of Ang II in male wild-type versus Noxa1-/- mice. Ang II increased epithelial Na+ channel (ENaC) levels and activation in the collecting duct principal epithelial cells of wild-type but not Noxa1-/- mice. Aldosterone induced ROS levels and Noxa1 and Scnn1a expression and ENaC activity in a mouse renal epithelial cell line, responses abolished by Noxa1 small-interfering RNA. Innovation and Conclusion: Ang II activation of renal NOXA1/NOX1-dependent ROS enhances tubular ENaC expression and Na+ reabsorption, leading to increased BP. Attenuation of AIH in females is attributed to weaker NOXA1/NOX1-dependent ROS signaling and efficient natriuresis. Antioxid. Redox Signal. 36, 550-566.

Marine Brominated Tyrosine Alkaloids as Promising Inhibitors of SARS-CoV-2.

There have been more than 150 million confirmed cases of SARS-CoV-2 since the beginning of the pandemic in 2019. By June 2021, the mortality from such infections approached 3.9 million people. Despite the availability of a number of vaccines which provide protection against this virus, the evolution of new viral variants, inconsistent availability of the vaccine around the world, and vaccine hesitancy, in some countries, makes it unreasonable to rely on mass vaccination alone to combat this pandemic. Consequently, much effort is directed to identifying potential antiviral treatments. Marine brominated tyrosine alkaloids are recognized to have antiviral potential. We test here the antiviral capacity of fourteen marine brominated tyrosine alkaloids against five different target proteins from SARS-CoV-2, including main protease (Mpro) (PDB ID: 6lu7), spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and non-structural protein 10 (nsp10) (PDB ID: 6W4H). These marine alkaloids, particularly the hexabrominated compound, fistularin-3, shows promising docking interactions with predicted binding affinities (S-score = -7.78, -7.65, -6.39, -6.28, -8.84 Kcal/mol) for the main protease (Mpro) (PDB ID: 6lu7), spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and non-structural protein 10 (nsp10) (PDB ID: 6W4H), respectively, where it forms better interactions with the protein pockets than the native interaction. It also shows promising molecular dynamics, pharmacokinetics, and toxicity profiles. As such, further exploration of the antiviral properties of fistularin-3 against SARS-CoV-2 is merited.

Stimulation of the Epithelial Na+ Channel in Renal Principal Cells by Gs-Coupled Designer Receptors Exclusively Activated by Designer Drugs.

The activity of the Epithelial Na+ Channel (ENaC) in renal principal cells (PC) fine-tunes sodium excretion and consequently, affects blood pressure. The Gs-adenylyl cyclase-cAMP signal transduction pathway is believed to play a central role in the normal control of ENaC activity in PCs. The current study quantifies the importance of this signaling pathway to the regulation of ENaC activity in vivo using a knock-in mouse that has conditional expression of Gs-DREADD (designer receptors exclusively activated by designer drugs; GsD) in renal PCs. The GsD mouse also contains a cAMP response element-luciferase reporter transgene for non-invasive bioluminescence monitoring of cAMP signaling. Clozapine N-oxide (CNO) was used to selectively and temporally stimulate GsD. Treatment with CNO significantly increased luciferase bioluminescence in the kidneys of PC-specific GsD but not control mice. CNO also significantly increased the activity of ENaC in principal cells in PC-specific GsD mice compared to untreated knock-in mice and CNO treated littermate controls. The cell permeable cAMP analog, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, significantly increased the activity and expression in the plasma membrane of recombinant ENaC expressed in CHO and COS-7 cells, respectively. Treatment of PC-specific GsD mice with CNO rapidly and significantly decreased urinary Na+ excretion compared to untreated PC-specific GsD mice and treated littermate controls. This decrease in Na+ excretion in response to CNO in PC-specific GsD mice was similar in magnitude and timing as that induced by the selective vasopressin receptor 2 agonist, desmopressin, in wild type mice. These findings demonstrate for the first time that targeted activation of Gs signaling exclusively in PCs is sufficient to increase ENaC activity and decrease dependent urinary Na+ excretion in live animals.

Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling.

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

Mechanisms and consequences of casein kinase II and ankyrin-3 regulation of the epithelial Na+ channel.

Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical "anchor" ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in ß-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a "switch" that favors Ank-3 binding to increase channel activity.

Comprehensive Virtual Screening of the Antiviral Potentialities of Marine Polycyclic Guanidine Alkaloids against SARS-CoV-2 (COVID-19).

The huge global expansion of the COVID-19 pandemic caused by the novel SARS-corona virus-2 is an extraordinary public health emergency. The unavailability of specific treatment against SARS-CoV-2 infection necessitates the focus of all scientists in this direction. The reported antiviral activities of guanidine alkaloids encouraged us to run a comprehensive in silico binding affinity of fifteen guanidine alkaloids against five different proteins of SARS-CoV-2, which we investigated. The investigated proteins are COVID-19 main protease (Mpro) (PDB ID: 6lu7), spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and a non-structural protein (nsp10) (PDB ID: 6W4H). The binding energies for all tested compounds indicated promising binding affinities. A noticeable superiority for the pentacyclic alkaloids particularly, crambescidin 786 (5) and crambescidin 826 (13) has been observed. Compound 5 exhibited very good binding affinities against Mpro (ΔG = -8.05 kcal/mol), nucleocapsid phosphoprotein (ΔG = -6.49 kcal/mol), and nsp10 (ΔG = -9.06 kcal/mol). Compound 13 showed promising binding affinities against Mpro (ΔG = -7.99 kcal/mol), spike glycoproteins (ΔG = -6.95 kcal/mol), and nucleocapsid phosphoprotein (ΔG = -8.01 kcal/mol). Such promising activities might be attributed to the long ω-fatty acid chain, which may play a vital role in binding within the active sites. The correlation of c Log P with free binding energies has been calculated. Furthermore, the SAR of the active compounds has been clarified. The Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) studies were carried out in silico for the 15 compounds; most examined compounds showed optimal to good range levels of ADMET aqueous solubility, intestinal absorption and being unable to pass blood brain barrier (BBB), non-inhibitors of CYP2D6, non-hepatotoxic, and bind plasma protein with a percentage less than 90%. The toxicity of the tested compounds was screened in silico against five models (FDA rodent carcinogenicity, carcinogenic potency TD50, rat maximum tolerated dose, rat oral LD50, and rat chronic lowest observed adverse effect level (LOAEL)). All compounds showed expected low toxicity against the tested models. Molecular dynamic (MD) simulations were also carried out to confirm the stable binding interactions of the most promising compounds, 5 and 13, with their targets. In conclusion, the examined 15 alkaloids specially 5 and 13 showed promising docking, ADMET, toxicity and MD results which open the door for further investigations for them against SARS-CoV-2.

High salt intake induces collecting duct HDAC1-dependent NO signaling.

We reported that high salt (HS) intake stimulates renal collecting duct (CD) endothelin (ET) type B receptor (ETBR)/nitric oxide (NO) synthase 1ß (NOS1ß)-dependent NO production inhibiting the epithelial sodium channel (ENaC) promoting natriuresis. However, the mechanism underlying the HS-induced increase of NO production is unclear. Histone deacetylase 1 (HDAC1) responds to increased fluid flow, as can occur in the CD during HS intake. The renal inner medulla (IM), in particular the IMCD, has the highest NOS1 activity within the kidney. Hence, we hypothesized that HS intake provokes HDAC1 activation of NO production in the IM. HS intake for 1 wk significantly increased HDAC1 abundance in the IM. Ex vivo treatment of dissociated IM from HS-fed mice with a selective HDAC1 inhibitor (MS-275) decreased NO production with no change in ET-1 peptide or mRNA levels. We further investigated the role of the ET-1/ETBR/NOS1ß signaling pathway with chronic ETBR blockade (A-192621). Although NO was decreased and ET-1 levels were elevated in the dissociated IM from HS-fed mice treated with A-192621, ex vivo MS-275 did not further change NO or ET-1 levels suggesting that HDAC1-mediated NO production is regulated at the level or downstream of ETBR activation. In split-open CDs from HS-fed mice, patch clamp analysis revealed significantly higher ENaC activity after MS-275 pretreatment, which was abrogated by an exogenous NO donor. Moreover, flow-induced increases in mIMCD-3 cell NO production were blunted by HDAC1 or calcium inhibition. Taken together, these findings indicate that HS intake induces HDAC1-dependent activation of the ETBR/NO pathway contributing to the natriuretic response.

Advances in venomics: Modern separation techniques and mass spectrometry.

Snake venoms are complex chemical mixtures of biologically active proteins and non-protein components. Toxins have a wide range of targets and effects to include ion channels and membrane receptors, and platelet aggregation and platelet plug formation. Toxins target these effectors and effects at high affinity and selectivity. From a pharmacological perspective, snake venom compounds are a valuable resource for drug discovery and development. However, a major challenge to drug discovery using snake venoms is isolating and analyzing the bioactive proteins and peptides in these complex mixtures. Getting molecular information from complex mixtures such as snake venoms requires proteomic analyses, generally combined with transcriptomic analyses of venom glands. The present review summarizes current knowledge and highlights important recent advances in venomics with special emphasis on contemporary separation techniques and bioinformatics that have begun to elaborate the complexity of snake venoms. Several analytical techniques such as two-dimensional gel electrophoresis, RP-HPLC, size exclusion chromatography, ion exchange chromatography, MALDI-TOF-MS, and LC-ESI-QTOF-MS have been employed in this regard. The improvement of separation approaches such as multidimensional-HPLC, 2D-electrophoresis coupled to soft-ionization (MALDI and ESI) mass spectrometry has been critical to obtain an accurate picture of the startling complexity of venoms. In the case of bioinformatics, a variety of software tools such as PEAKS also has been used successfully. Such information gleaned from venomics is important to both predicting and resolving the biological activity of the active components of venoms, which in turn is key for the development of new drugs based on these venom components.

Cisplatin Decreases ENaC Activity Contributing to Renal Salt Wasting Syndrome.

Cisplatin (CDDP) is an important anticancer drug. A common side effect of CDDP is renal salt and water-wasting syndrome (RSWS). The origin of RSWS is obscure. Emerging evidence, though, suggests that broad inhibition of sodium transport proteins by CDDP may result in decreases in tubular reabsorption, causing increases in sodium and water excretion. In this sense, CDDP would be acting like a diuretic. The effect of CDDP on the epithelial Na+ channel (ENaC), which is the final arbiter fine-tuning renal Na+ excretion, is unknown. We test here whether CDDP affects ENaC to promote renal salt and water excretion. The effects of CDDP and benzamil (BZM), a blocker of ENaC, on excretion of a sodium load were quantified. Similar to BZM, CDDP facilitated renal Na+ excretion. To directly quantify the effects on ENaC, principal cells in split-open tubules were patch clamped. CDDP, at doses comparable to those used for chemotherapy (1.5 µM), significantly decreased ENaC activity in native tubules. To further elaborate on this mechanism, the dose-dependent effects of CDDP on mouse ENaC (mENaC) heterologously expressed in Chinese Hamster Ovary (CHO) cells were tested using patch clamping. As in native tubules, CDDP significantly decreased the activity of mENaC expressed in CHO cells. Dose-response curves and competition with amiloride identified CDDP as a weak inhibitor of ENaC (apparent IC50 = 1 µM) that competes with amiloride for inhibition of the channel, weakening the inhibitory actions of the latter. Such observations are consistent with CDDP being a partial modulator of ENaC, which possibly has a binding site that overlaps with that of amiloride. These findings are consistent with inhibition of ENaC by CDDP contributing to the RSWS caused by this important chemotherapy drug.

Recent progress and challenges in drug development against COVID-19 coronavirus (SARS-CoV-2) - an update on the status.

Coronaviruses are a large group of viruses known to cause illnesses that vary between the common cold and more severe diseases to include severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). A novel coronavirus was identified in December 2019 in Wuhan city, Hubei province, China. This virus represents a new strain that has not been previously identified in humans. The virus is now known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resulting disease is called coronavirus disease 2019 (COVID-19). The World Health Organization (WHO) declared the novel coronavirus outbreak a global pandemic in March 2020. Despite rigorous global containment and quarantine efforts, the incidence of COVID-19 continues to rise, with more than 1,948,617 laboratory-confirmed cases and over 121,846 deaths worldwide. Currently, no specific medication is recommended to treat COVID-19 patients. However, governments and pharmaceutical companies are struggling to quickly find an effective drug to defeat the coronavirus. In the current review, we summarize the existing state of knowledge about COVID-19, available medications, and treatment options. Favilavir is an antiviral drug that is approved in Japan for common influenza treatment and is now approved to treat symptoms of COVID-19 in China. Moreover, Chloroquine and hydroxychloroquine, drugs used to treat malaria and arthritis, respectively, were recommended by the National Health Commission of the People's Republic of China for treatment of COVID-19. Presently, chloroquine and hydroxychloroquine are under investigation by the US Food and Drug Administration (FDA) as a treatment for COVID-19. The first COVID-19 vaccine is not expected to be ready for clinical trials before the end of the year.

Phosphatidylinositol 4,5-bisphosphate directly interacts with the ß and γ subunits of the sodium channel ENaC.

The plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of diverse ion channels to include the epithelial Na+ channel ENaC. Whether PIP2 regulation of ENaC is due to a direct phospholipid-protein interaction, remains obscure. To date, possible interaction of PIP2 with ENaC primarily has been tested indirectly through assays of channel function. A fragment-based biochemical analysis approach is used here to directly quantify possible PIP2-ENaC interactions. We find using the CIBN-CRY2 optogenetic dimerization system that the phosphoryl group positioned at carbon 5 of PIP2 is necessary for interaction with ENaC. Previous studies have implicated conserved basic residues in the cytosolic portions of ß- and γ-ENaC subunits as being important for PIP2-ENaC interactions. To test this, we used synthetic peptides of these regions of ß- and γ-ENaC. Steady-state intrinsic fluorescence spectroscopy demonstrated that phosphoinositides change the local conformation of the N terminus of ß-ENaC, and two sites of γ-ENaC adjacent to the plasma membrane, suggesting direct interactions of PIP2 with these three regions. Microscale thermophoresis elaborated PIP2 interactions with the N termini of ß- (Kd ∼5.2 µm) and γ-ENaC (Kd ∼13 µm). A weaker interaction site within the carboxyl terminus of γ-ENaC (Kd ∼800 µm) was also observed. These results support that PIP2 regulates ENaC activity by directly interacting with at least three distinct regions within the cytoplasmic domains of the channel that contain conserved basic residues. These interactions are probably electrostatic in nature, and are likely to bear a key structural role in support of channel activity.

Cerebellar Ataxia Caused by Type II Unipolar Brush Cell Dysfunction in the Asic5 Knockout Mouse.

Unipolar brush cells (UBCs) are excitatory granular layer interneurons in the vestibulocerebellum. Here we assessed motor coordination and balance to investigate if deletion of acid-sensing ion channel 5 (Asic5), which is richly expressed in type II UBCs, is sufficient to cause ataxia. The possible cellular mechanism underpinning ataxia in this global Asic5 knockout model was elaborated using brain slice electrophysiology. Asic5 deletion impaired motor performance and decreased intrinsic UBC excitability, reducing spontaneous action potential firing by slowing maximum depolarization rate. Reduced intrinsic excitability in UBCs was partially compensated by suppression of the magnitude and duration of delayed hyperpolarizing K+ currents triggered by glutamate. Glutamate typically stimulates burst firing subsequent to this hyperpolarization in normal type II UBCs. Burst firing frequency was elevated in knockout type II UBCs because it was initiated from a more depolarized potential compared to normal cells. Findings indicate that Asic5 is important for type II UBC activity and that loss of Asic5 contributes to impaired movement, likely, at least in part, due to altered temporal processing of vestibular input.

Comparative study of the in vivo toxicity and pathophysiology of envenomation by three medically important Egyptian snake venoms.

Snakebite envenomation is a serious medical problem in many developing tropical and subtropical countries. Envenomation is registered by the World Health Organization as a neglected tropical disease due to critical shortages in the production of antivenom. Envenomation causes more than 100,000 deaths annually. Snakebites result in several effects to include edema, blistering, hemorrhage, necrosis and respiratory paralysis. Antivenom is the preferred treatment for the systemic effects of snakebite envenomation, though these are often ineffective in neutralizing venom toxin-induced local tissue damage. To effectively treat snakebites, it is important to determine the lethal potency and pathophysiological effects induced by specific snake venoms. In the current study, we compared the lethality, and the hemorrhagic and dermonecrotic activities of venoms from three snakes in Egypt that are the primary causes of local tissue necrosis. Our data show that the intraperitoneal median lethal doses (LD50) for Cerastes cerastes, Echis carinatus and Naja nigricollis venoms are 0.946, 1.744 and 0.341 mg/kg mouse body weight, respectively. These results indicated that N. nigricollis venom is the most toxic and significantly accelerated the time of death compared to the other two venoms. However, no hematoma or associated edema appeared upon sub-plantar injection of N. nigricollis venom into the mice hind paw. Two hours following intradermal injection of C. cerastes and E. carinatus venoms, macroscopic analysis of the inner surface of mouse skin showed severe hemorrhagic lesions, whereas only insignificant hemorrhagic lesion appeared in mice injected with the highest dose of N. nigricollis venom. Furthermore, the minimum necrotic doses (MND) for the same venoms were 43.15, and 70.87 µg/mouse, or not observed in the case of N. nigricollis venom, respectively. These LD50 values and pathophysiological results can be used to guide development of antivenom against bites by these dangerous Egyptian snakes.