bluuebunny/crossref_metadata_2025_split huggingface dataset:
ASIC1a/α‐ENaC Hybrid Channels Contribute to Alveolar Fluid Clearance
Authors: Trac Phi , Greenlee Megan M , Al‐Khalili Otor , Eaton Douglas C 
Published: November 2023

Pneumonia affects 450 million people globally per year with about 4 million deaths. Pneumonia mortality is caused by abnormal alveolar fluid clearance (AFC) preventing normal gas exchange with consequent hypoxemia. Since knockout of α‐ENaC completely prevents new born mice from clearing fluid from their lungs, it is reasonable to conclude that α‐ENaC is responsible for AFC; but if all Na+ reabsorption was via ENaC alone, then amiloride should block all AFC, but it blocks only about half. Single channel measurements from alveolar epithelial cells show why. There is a channel with properties like ENaC expressed in heterologous expression systems, but there is another channel present at almost the same frequency that is non‐selective for Na+ over K+, has a larger conductance, and shorter mean open and closed times. These two types of channels have been called highly selective channels (HSC) and non‐selective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC channel number. The composition of NSC channels is unknown, but appears to contain α‐ENaC since knocking down this subunit reduces the number of NSC channels (knocking down β‐ or γ‐ENaC has no effect on NSC number). We now hypothesize that NSC channels consist of at least one α‐ENaC subunit and one or more ASIC1a protein subunits (Acid‐Sensing Ion Channel 1a). ASIC1a is another member of the ENaC/degenerin protein family that is strongly expressed in alveolar epithelial cells. We used molecular biological methods to manipulate the amount of ASIC1a and α‐ENaC in heterologous expression systems and alveolar cells and assessed the effect on HSC and NSC channels using single channel measurements. ASIC1a and α‐ENaC co‐immunoprecipitate from lung cell lysates and are closely associated in mammalian two‐hybrid assays. ASIC1a/α‐ENaC coexpression in FRT cells produces NSC channels that are very similar to NSC channels in mouse lung cells. shRNA knock‐down of ASIC1 in a lung cell line reduces the frequency of NSC channels from 91 % to 10 % of patches with no effect on HSC channels. α‐ENaC silencing reduced HSC frequency from 55% to 8% of patches and NSC channels from 86% to 12% of patches. The frequency of NSC in patches from lung slices is also reduced in mice instilled with ASIC1a shRNA silencing vectors. Therefore, we conclude that NSC channels are ASIC1a/α‐ENaC hybrid channels that contribute to maintaining AFC. The regulation of NSC and HSC channels is so different that it may require a new paradigm to explain normal AFC and AFC in the face of a viral challenge.Support or Funding InformationR37‐DK037963 to DCE; APS/NHLBI STRIDE to PT
Regulation of ENaC by specific palmitoyltransferases
Authors: Mukherjee Anindit , Wong Zhijian , Poland Paul A , Montalbetti Nicolas , Butterworth Michael , Fukata Masaki , Kleyman Thomas R , Hughey Rebecca P 
Published: November 2023

The epithelial sodium channel (ENaC) is a key mediator of extracellular fluid volume homeostasis and blood pressure control. It is a member of the ENaC/degenerin family and is regulated by extracellular protons, chloride, sodium, and proteases. ENaC is also regulated by phosphorylation, ubiquitination, inositol phospholipids, and Cys‐palmitoylation. We have previously shown that mouse ENaC is palmitoylated at Cys residues in the β(C43, C557) and γ(C33, C41) subunit cytoplasmic tails. ENaCs lacking β and/or γ subunit palmitoylation had normal surface expression but reduced open probability. A family of enzymes, referred to as DHHCs based on their conserved Asp‐His‐His‐Cys (DHHC) catalytic motif, catalyzes Cys‐palmitoylation. The 23 mouse DHHCs were individually co‐expressed in Xenopus oocytes with either wild‐type ENaC, or ENaC with a mutant β and/or mutant γ subunit lacking Cys palmitoylation sites. Five of the 23 DHHCs activated ENaC. Both the β and γ subunit palmitoylation sites were required for full ENaC activation by DHHCs. As the five activating DHHCs are expressed in mouse CCD cells as evidenced by RT‐PCR, we tested whether inhibiting DHHC activity with the irreversible palmitoylation inhibitor, 2‐bromopalmitate (2‐BP) altered ENaC activity. Short circuit currents (Isc) were measured in mCCD cells. Isc began to fall within 5 min after addition of apical 2‐BP (25 μM), and reached a steady state after 30 minutes (50.1 ± 2.8 mA/cm2 (vehicle control) vs. 11.3 ± 1.2 mA/cm2 (2‐BP) (N=9, p<0.0001). Conserved cytoplasmic His‐Gly (HG) tracts that regulate ENaC gating are in close proximity to both βC43 and γC41. Mutations of the HG tract have been shown to decrease ENaC activity and open probability. We examined whether mutating the HG tract affects ENaC activation by Cys‐palmitoylation. While DHHC2 increased activity of wild type ENaC, channels with a βH36A mutation were not activated by DHHC2. These data suggest that an intact HG tract is required for channel activation by Cys‐palmitoylation.Support or Funding InformationDK051391, P30‐DK079307
Mitochondrial Transcription Factor A: The root to cardiomyocte survival
Authors: Kunkel George H. , Chaturvedi Pankaj , Familtseva Anastasia , Tyagi Suresh C. 
Published: November 2023

Mitochondrial Transcription Factor A (TFAM), an essential component of mitochondrial biogenesis, is lost in heart failure (HF) models. Functionally, TFAM encapsulates mitochondrial DNA (mtDNA) into a mitochondrial nucleoid structure, creating a safe environment for mtDNA processes such as transcription, translation, and repair. Importantly, this environment contains molecules, which reduce oxidative stress (ROS) to mtDNA. We hypothesize that TFAM regulates mitochondrial oxidative stress through Lon protease and modulates Serca2a expression. To test this hypothesis, exogenous TFAM treatment was administered to HL‐1 cardiomyocytes with and without H2O2. Experimental analysis of a ROS‐driven Lon protease, TFAM and Serca2a were observed. Interestingly, under oxidative stress the mRNA product of TFAM was stable, as shown in RT‐PCR, while western blotting showed that TFAM protein was lost. Additionally, confocal imaging of TFAM and Lon in the presence of oxidative stress provided further evidence for this negative correlation. Therefore, a protease is degrading TFAM during increased levels of ROS, as seen in heart failure model studies. Lon protease is not found within the nucleus but is highly expressed under H2O2 treatment. Serca2a, an SR calcium storage transporter was enhanced by TFAM treatment. In conclusion, our results suggest that a loss of TFAM correlates with a loss of Serca2a and an increase in Lon protease, which subsequently lead to high oxidative stress, calcium mishandling, and cardiac dysfunction.Support or Funding InformationNIH Grants: HL‐74185 & HL‐108621
Knockdown of the Expression of Gamma‐Adducin in the Renal Afferent Arteriole Enhances BK Channel Activity
Authors: Fan Fan , Ge Ying , Pabbidi Mallikarjuna R , Harder David R , Roman Richard J 
Published: November 2023

We recently identified a region on chromosome 1 containing 15 genes including Gamma‐Adducin (Add3) that causes the impaired myogenic response in the renal afferent arteriole (Af‐Art) and the development of renal vascular injury in Fawn Hood Hypertensive (FHH) rats. To determine which isoform of Add3 is expressed in the vasculature and whether Add3 is the viable candidate gene that plays a role in the modulation of myogenic response in renal microvessels, we knocked down the expression of Add3 with newly designed 27‐mer Dicer‐Substrate RNAi (DsiRNA) in rat Af‐art cultured for 36 hours. We then identified positively transfected vascular smooth muscle cells (VSMCs) using a red fluorescent indicator, and examined the large conductance calcium sensitive potassium (BK) current using patch clamp techniques. Both Add3 isoforms were detected in brain, kidney and liver, however, only isoform 2 was detected in the vasculature. The expression of Add3 was reduced in the renal microvessels and in the membrane fraction of the brain of FHH vs. FHH.1BN rats. Add3 DsiRNA completely blocked the expression of Add3 protein in 293 cells and reduced the expression of mRNA in a dose‐dependent manner up to 50% in the cultured Af‐Art. The inner diameters of the Af‐Art transfected with Add3 DsiRNA dilated by 4 ± 3 % in response to an elevation in transmural pressure from 60 to 120 mmHg. In contrast, these vessels still constricted normally by 10 ± 1 % in vessels transfected with scrambled siRNA. The myogenic response of Af‐Art isolated from our novel Add3 KO rats is impaired versus controls. BK current was 3‐fold higher in VSMC freshly isolated from Af‐Art that were transfected with Add3 DsiRNA as indicated by the appearance of siGLO red fluorescence compared with that seen in non‐transfected cells. Similarly, BK current was higher in 293BKα cells transfected with Add3 DsiRNA or in VSMC isolated from Af‐Art of FHH relative to control rats and was blocked by IBTX. BKα protein expression was greater in the membrane fraction in FHH vs. FHH.1BN rats. It appeared a stronger florescence at the membrane of 293BKα cells transfected with Add3 DsiRNA when staining with BKα implying an inhibition of endocytosis of BKα from the cell membrane. These results indicate that vasculature specifically expressed Add3 isoform 2, knockdown of the expression of Add3 impairs the myogenic response of Af‐Art that is similar as seen in FHH rats and associated with an elevation in BK channel activity. It supports the hypothesis that Add3 is a viable candidate gene that may play a causal role in the impaired myogenic response of the renal circulation in FHH rats and contributes to the development of renal injury.Support or Funding InformationThis study was supported by grants HL36279 and DK104184 (RJR) from the National Institutes of Health.
Potassium Channels in Ischemic Stroke
Authors: Chen Yi‐Je (Jay) , Nguyen Hai M , O'Donnell Martha E. , Wulff Heike 
Published: November 2023

On average, a stroke occurs every 40s in the United States and 87% of these cases are ischemic strokes. In the acute phase, cerebral edema is a major cause of neuronal death, and then activated microglia/macrophages significantly contribute to the secondary inflammatory damage later in ischemic stroke. Neither the physiological or pathological roles of potassium channels in ischemic stroke have been systematically explored. Our studies reveal that potassium channels are important in cerebral edema formation and microglia/macrophage activation. KCa3.1, a calcium‐activated potassium channel, physiologically regulates ion and fluid secretion in the lung and gastrointestinal tract. It is also expressed on vascular endothelium where it participates in blood pressure regulation. Our recent study of the expression and physiological role of KCa3.1 in blood–brain barrier (BBB) endothelium found that BBB endothelial cells exhibit KCa3.1 protein and activity facilitating Na+ transportation from the blood into the brain transcellularly through the co‐operation of multiple cotransporters, exchangers, pumps, and channels in normal physiological conditions and the early stages of cerebral ischemia, when the BBB is still intact. Pharmacological blockade with KCa3.1 blocker, TRAM‐34, significantly reduced Na+ uptake, and cytotoxic edema measured by MRI in the ischemic rat brain with permanent middle cerebral artery occlusion (MCAO) in the first 3 hours of ischemic stroke. (Chen et el. 2015 Stroke 46:237.) Together with the voltage‐gated potassium channel Kv1.3, KCa3.1 also regulates membrane potential and Ca2+ signaling in microglia and monocyte‐derived macrophages. We recently evaluated acute isolated microglia/macrophage from mice subjected to MCAO with eight days of reperfusion. Microglia from the infarcted area exhibited higher densities of K+ currents with the biophysical and pharmacological properties of Kv1.3, KCa3.1 and Kir2.1 than microglia from non‐infarcted control brains. Similarly, immunohistochemistry on human infarcts showed strong Kv1.3 and KCa3.1 immunoreactivity on activated microglia/macrophages. Pharmacological inhibition or genetic deletion of KCa3.1 resulted in significantly smaller infarct areas on day‐8 after MCAO and improved neurological deficits. Both manipulations reduced microglia/macrophage activation and brain cytokine levels. (Chen et al. 2015 JCBFM; epub) In summary, K+ channels can be a potential pharmacological target for ischemic stroke to slow down cerebral edema formation and reduce the secondary inflammatory damage from activated microglia/macrophage.Support or Funding InformationSupported by AHA & NIHGM
Selective Knockout of Astrocytic Na+/H+ Exchanger Isoform 1 is Neuroprotective in Ischemic Stroke Injury
Authors: Begum Gulnaz , Wang Shaoxia , Zhao Hanshu , Bhuiyan Mohammad Iqbal Hossain , Nepomuceno Rachel , Ye Qing , Stolz Donna Beer , Sun Ming , Chen Yinhuai , Shull Gary E , Dandan Sun 
Published: November 2023

We previously reported that Na+/H+ exchanger isoform 1 (NHE1) plays a role in the dysregulation of ionic homeostasis in astrocytes under ischemic conditions. In this study, we created an Nhe1flox/flox mouse line (Nhe1f/f) with exon 5 of Nhe1 flanked with two loxP sites in order to selectively ablate NHE1 in astrocytes when crossed with cell‐type specific GFAPCreERT2 Cre‐recombinase mouse line. GfapCreER/+;NHE1f/f mice at P60–90 were treated with either tamoxifen (Tam, 75mg/kg/day, i.p.) or corn oil for 5 days to induce Cre recombination. 30 days post injection, mice underwent a transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. With ultrastructural and immunocytochemical analyses at 2 days post‐tMCAO, we detected perivascular reactive astrocyte formation, extensive perivascular edema, swollen and vacuolated endothelial cells and damaged tight junctions in the GfapCreER/+;NHE1f/f mice treated with the corn oil, which was accompanied with AQP4 and laminin accumulation. In contrast, reduced AQP4 protein expression and less laminin accumulation were detected in the microvessels of the Tam‐treated GfapCreER/+;NHE1f/f mice. Selective knockout of NHE1 in GFAP+ astrocytes also prevented reactive astrocyte formation, decreased S100β release, and reduced microvessel damage and the BBB leakage after ischemic stroke. The GfapCreER/+;Nhe1f/f mice treated with Tam exhibited smaller ischemic infarct and less pronounced neurological function deficits. Taken together, this is the first study to report that astrocytic NHE1 plays an important role in reactive astrocyte‐mediated ischemic brain damage.Support or Funding InformationNIH R01 NS048216 (D.S)
Iron Effect On Erythrocyte Na,K‐Atpase Activity
Authors: Barbosa Leandro A , Fernandes Tamara , Sousa Leilismara , Cortes Vanessa F , Santos Herica L 
Published: November 2023

Iron is a chemical element essential to humans; however, its overhead can be toxic to the body, because it can mediate events of lipid peroxidation, stimulate the formation of collagen and produce reactive oxygen species (ROS). The objective of this work was evaluate the modulation of iron on erythrocyte Na,K‐ATPase activity. Red blood cells (RBC) were collected from 24 patients with iron overload forwarded to the realization of therapeutic phlebotomy. We assessed the erythrocyte Na,K‐ATPase activity, TBARS levels, total cholesterol and phospholipid of these patients, and 14 subjects were recruited to form the control group. Furthermore we incubated RBC from our control group with 0,8 nM H2O2 and 1 μM FeCl3 for 24h and evaluate the same parameters and to evaluate the kinetics of Na,K‐ATPase activation. The activity of the Na,K‐ATPase increased by 327% for patients compared to the control group. Also, the levels of TBARS were increased by 446% and the levels of total cholesterol and phospholipids were decreased. In order to verify if these effects were modulated by iron, we incubated the RBCs with 0,8 nM H2O2 and 1 μM FeCl3 for 24h. After incubation with 1 μM FeCl3 the Na,K‐ATPase activity were activate by 132%, but H2O2 not caused any effect. Moreover, only FeCl3 was able to modulate the decrease of total cholesterol and phospholipids levels, and neither H2O2 nor FeCl3 caused modifications on TBARS levels. Moreover, iron treatment of erythrocyte for 24h caused modifications to the affinity of the Na,K‐ATPase for sodium and potassium, but the direct treatment of iron on membrane preparations caused inhibition of the Na,K‐ATPase, suggesting that the iron modulates the erythrocyte to change the Na,K‐ATPase.Our data indicate that iron overload induces increased activity of the Na,K‐ATPase in erythrocytes and can be harmful to the cell, due to the high energy consumption, thus modulating the functioning of other enzymes. The increased of ROS can be involved in this effect, but probable the cell has more antioxidants defenses against H2O2 than oxidative damage modulated by iron overload.Support or Funding InformationFAPEMIG, CNPq, CAPES, HEMOMINAS
Stimulated Transepithelial Ion Flux in a Choroid Plexus Epithelial Cell Line
Authors: Ahmed Shehab , Otun Ayodamola , Schwerk Christian , Schroten Horst , Blazer‐Yost Bonnie L. 
Published: November 2023

Production and maintenance of the cerebral spinal fluid (CSF) is crucial for brain health and normal function. Alterations in normal homeostatic balance can occur in response a variety of pathological etiologies including traumatic brain injury, stroke, infection and cancerous lesions. Overproduction or blockage of CSF flow can also lead to hydrocephalus in both the pediatric population and adults. These alterations are remarkably difficult to treat and, in many cases, surgery is the only option. The choroid plexus, an epithelial cell lined tuft of capillaries that project into the cerebral ventricles, is responsible for a substantial portion of the CSF production. The capillaries of the CP are relatively leaky while the epithelial cells surrounding this vasculature form the blood‐CSF barrier and control the amount and composition of the CSF. A stable porcine choroid plexus epithelial cell line (PCP‐R) with a high transepithelial resistance (TEER) has been developed. We used Ussing style electrophysiology to characterize stimulated transepithelial ion transport measured as short circuit current (SCC) in confluent PCP‐R cells. In response to forskolin, a compound known to activate adenylyl cyclase and increase intracellular cAMP, there is a sustained increase in transepithelial ion flux that is consistent with an increase in cation absorption and/or anion secretion. This stimulated transport is partially inhibited by 1 mM BaCl2, used as a pan inhibitor of K+ channels. It is also partially inhibited by 10 mM T16Ainh‐A01, an inhibitor of the Ca2+ activated Cl− channel TMEM16a and by 50 nM GLY H101, an inhibitor of the cAMP‐stimulated Cl− channel CFTR. Neither the stimulatory nor inhibitory responses were accompanied by a substantial change in TEER. An agonist of the transient receptor potential vanilloid 4 (TRPV4) cation channel, GSK1016790, elicits a prolonged ion transport response that is the opposite of the forskolin stimulated response. The TRPV4 agonist elicits an increase in cation secretion and/or anion absorption. Interestingly, this response to the agonist is accompanied by a reversible decrease in TEER (>50%). The effect of the agonist on both SCC and TEER can be blocked by pretreatment with HC067047, a TRPV4 antagonist. If added after agonist stimulation, the TRPV4 antagonist can also fully reverse the agonist stimulated ion flux and can substantially restore the agonist stimulated decrease in TEER. Studies such as these are important for characterizing the factors that may disturb the normal CSF production and lead to an accumulation of CSF that can, over time, cause cellular loss and brain injury.Support or Funding InformationThis research was supported by an Indiana University Collaborative Research Grant fund of the Office of the Vice President for Research, and by a grant from the Vice Chancellor's Office, IUPUI.
Electrophilic activation of the nociceptive channel TRPA1 requires a 6000‐fold increase in reactivity at cysteine 621 that is dependent on a neighboring lysine
Authors: Bahia Parmvir , Parks Thomas , Stevens Stanley , Taylor‐Clark Thomas 
Published: November 2023

Mammalian TRPA1 is a cation channel expressed on nociceptive sensory nerves and contributes to pain/nociceptive responses to oxidative stress and inflammation. In particular TRPA1 is rapidly activated by oxidative stress‐associated electrophiles capable of covalent modification of cysteine residues. Putative ‘functionally electrophile‐sensitive’ Cys‐containing proteins must be put into context of antioxidant microenvironments containing mM glutathione and enzymes that accelerate electrophilic‐GSH reactions. Thus the mechanisms underlying the rapid reaction rates of TRPA1 Cys are fundamental to its ability to act as a competent sensor of oxidative stress and electrophilic damage. We have used physiologically relevant concentrations and exposures in both binding studies and functional experiments to offer mechanistic insights into the functional responses of TRPA1 to electrophiles. Human and mouse TRPA1 are rapidly covalently modified by electrophiles and this response is insensitive to TRPA1 inhibition or the absence of polyphosphates. Orthologs and paralogs insensitive to electrophiles display no rapid binding. Mass spectroscopy studies identified only 4 Cys rapidly modified: C273, C621, C665 and C1085. 98% of C621 were modified with 120s of 30uM iodoacetamide treatment. Only 3–4% of the other three Cys were modified. The second order reaction rate of C621 with iodoacetamide is >6000‐fold faster than typical Cys residues. The mutant C621A displays greatly reduced electrophilic binding and is not activated by iodoacetamide. Neighboring basic residues can increase thiol reactivity by promoting the thiolate state. Mutation of K620 eliminated electrophilic binding and electrophile‐induced activation, despite continued presence of C621. This data suggest that TRPA1 possesses a complex reactive Cys profile centering on the extraordinary reactivity of Cys 621, which is determined by its low pKa under the influence of its neighbor Lys 620. The reactivity of C621 is critical to TRPA1's functional sensitivity to oxidative stress. The extraordinary reactivity of TRPA1 C621 is likely fundamental to nociceptive responses to oxidative stress‐associated inflammation.Support or Funding InformationNHLBI R01HL119802 and R01HL119802‐S1