bluuebunny/crossref_metadata_2025 huggingface dataset:
Tight Junction Proteins In Adult Rainbow Trout Skin: Regional Distribution, Response To Environmental Salinity, And Potential Regulatory Mechanisms
Authors: Gauberg Julia , Kolosov Dennis , Kelly Scott P.
Published: November 2023

Freshwater (FW) fishes reside exist in aquatic habitats that are more dilute than their inner fluids. This leads to passive ion loss across tissues that directly interface with the surrounding medium, such as the gill and skin. The barrier properties of adult teleost fish skin in FW are well documented and in most cases, the trunk skin of adult fishes is void of any significant capacity for active ion movement. Therefore, despite its complex structure and extensive contact with the external environment, the skin of adult fishes is classically regarded as a relatively static, passive barrier to diffusional ion loss in FW. Nevertheless, recent observations have revealed the presence of a considerable number of genes encoding tight junction (TJ) proteins in the adult skin of several fish species. In addition, some studies have also shown that select TJ proteins in the skin of fish significantly alter in abundance upon exposure to environments that vary in ionic composition. This suggests that the molecular architecture of the skin TJ complex may play an important role in regulating skin permeability during environmental change. Despite this, almost nothing is known about how the molecular physiology of the TJ complex in this boundary tissue is regulated. Additionally, structural and functional differences are known to exist along the dorsoventral axis of the adult fish skin. However, none of the studies conducted to date, have taken this documented regional heterogeneity into account. This study examined TJ protein response to environmental change (acclimation to ion‐poor water, IPW) in freshwater (FW) rainbow trout (Oncorhynchus mykiss) skin. Changes in TJ protein abundance along the dorsoventral axis of the trout skin taken into consideration, as were possible roles for prolactin and cortisol, hormones involved in salinity‐dependent regulation of TJ/passive permeability. Differences in abundance of cldn‐3a, −5b, −6, −10c, −30, −32a and zo‐1 were detected along the dorsoventral axis of rainbow trout skin and many of these were found to be enriched in the lateral and dorsolateral regions. In addition, cldn mRNA abundance altered in response to IPW acclimation in a region‐specific manner. Osmoregulatory hormones, cortisol and prolactin, and their receptors were implicated in the regulation of Cldn transcript abundance in rainbow trout skin. These data provide a unique look at the region‐specific endocrine regulation of TJ complex components in adult fish skin.

Support or Funding Information

This work was funded by National Science and Engineering Research Council of Canada
Loss of the Tight Junction Protein Claudin 15 Causes Malabsorption of Peptide in Murine Intestine
Authors: Hayashi Hisayoshi , Tajima Haruna , Watanabe Miki , Ishizuka Noriko
Published: November 2023

It is known that the claudin family of tight junction proteins is critical in determining paracellular ionic permeability and selectivity. We have shown that loss of claudin 15 results in decreased luminal Na+ concentration and glucose malabsorption in the small intestine. To gain further insight into the relationship between intestinal Na+ metabolism and changes in nutrient absorption induced by the loss of claudin 15, we investigated the site of absorption of electrolytes and peptide in claudin 15 knockout (cldn15KO) mice and compared this with wild‐type mice under in vivo conditions. Mice were fed a diet supplemented with 14C‐polyethylene glycol (PEG) 4000 as a non‐absorbable marker and 3H‐Gly‐Sar (non‐hydrolyzable dipeptide). After feeding, the small intestine was isolated and divided into six segments, the luminal contents collected for analysis of Na+‐concentration and the level of 14C‐PEG4000 and 3H‐Gly‐Sar. Gastric emptying time, assessed by measuring 14C‐PEG4000, was not changed in cldn15KO compared to wild‐type mice. Total luminal contents in the small intestine were increased in cldn15KO mice and the sojourn time of digesta in the upper jejunum was increased 3‐fold compared with wild‐type mice. Robust Na+ secretion and rate of absorption were observed in the upper jejunum in wild‐type mice and this was attenuated in cldn15KO mice. Total amount of luminal Gly‐Sar were increased, while absorption rates of Gly‐Sar were decreased, in the upper jejunum of cldn15KO mice. This implies that decrease of expression level of pepT1 and /or NHE3. Therefore, quantitative real‐time PCR was used to determine the mRNA transcription of pepT1 and NHE3. However, there was no difference between cldn15KO and wild‐type mice. We next measured Gly‐Sar‐induced short‐circuit current (Isc) in Ussing chambers systems. Upon Gly‐Sar was added to the mucosal side in wild‐type mice, robust biphasic increment of Isc was observed and this was attenuated by mucosal addition of NHE3 inhibitor S3226. In cldn15KO mice, Gly‐Sar‐induced Isc was significantly decreased compared to wild‐type mice and was not sustained the plateau phase. These results suggest that NHE3 activation mechanism(s) induced by peptide absorption was not operated properly in cldn15KO mice.
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 Information

R37‐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 Information

DK051391, 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 Information

NIH 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 Information

This 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 Information

Supported 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 Information

NIH 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 Information

FAPEMIG, 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 Information

This 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 Information

NHLBI R01HL119802 and R01HL119802‐S1
Membrane channel gene expression in costal cartilage from patients with chest wall deformities
Authors: Asmar Anthony , Barrett‐Jolley Richard , Kelly Robert , Tanaz Royena , Stacey Michael
Published: November 2023

Chondrocytes form permanent cartilagein joints and ribs. Although much work has been undertaken on joint cartilage with respect to osteoarthritis, little is known about rib, or costal, cartilage. Inherited disorders of costal cartilage include pectus excavatum(PE) and pectus carinatum (PC), where the sternum is either depressed towards the spine, or protrudes outwardly forming a ‘pigeon chest’ respectively. In severe case there are profound consequences on heart and lung function. The occurrence of these disorders is approximately 1 in 400–1,000 with a strong male bias. Key to chondrocyte function is the ability to respond to mechanical loads with changes of metabolic activity. This mechano transduction property is, in part, mediated through the activity of a range of constitutively expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to other musculoskeletal diseases such as osteoarthritis and Albers‐Schönberg. In this study the objective was to measure consistency of gene expression between channelomes of chondrocytes from patients with chest wall deformities compared to apparentlynormal samples of costal cartilage obtained from individuals with no known history of chest wall deformity. Gene expression was also compared to myocardial cells with well‐defined channelome expression. In our preliminary data we found 23 genes for different ion channels commonly expressed between costal chondrocytes. Interestingly, there were five ion channel genes observed which are characteristic to chondrocytes from chest wall deformities and five observed only in control chondrocytes. mRNA expression of the gap junction/innexin‐like genes;Panx1, Cx43 and Cx45 was also detected. In conclusion, this data highlights similarities and differences between chondrocyte membrane channel gene expressions in cells derived from patients versus control cost alcartilage. Overall, the data shows high expression of a range of mechanically and metabolically sensitive membrane channels suggesting that chondrocytemechanotransduction may play an important role in chest wall deformity.

Support or Funding Information

Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institute of Health under the award number R21AR063334. The content is solely the responsibility of the authors and does not necessarily represent the views of the NIH. We also acknowledge partial support from the Breeden Adams Foundation of Norfolk, VA, USA.
Modulation of the BK Channel During Acute Spinal Cord Injury is Neuroprotective
Authors: JACOBSEN MARIANNE C , Lett Kristin A , Karnitsky Max , Arwini Balsam M , Barden John M , Buttigieg Josef
Published: November 2023

Spinal cord injury results in significant neuronal and glial cell death resulting in impaired neurological and motor function. While there are often surviving axonal tracts, these are frequently dysmyelinated or demyelinated. Key to this pathology is uncontrolled Ca2+ entry, resulting in excitotoxicity and cell death. We propose that by activating mechanisms attenuating global depolarization and limiting Ca2+ entry, we can reduce neuronal death and significantly improve neurological and motor function. To this effect, we previously demonstrated that the large‐conductance, voltage and Ca2+‐activated K+ (BK or Maxi K+) channel is altered after SCI, and may play a part in the neuropathology of SCI. In this study, we set out to explore whether the use of a BK channel activator, Isopimaric acid (ISO), is neuroprotective post spinal cord injury. Wister rats received a 25‐g clip compression injury at the T7 levels for 1 min. One hour post‐surgery, they were administered ISO (120 μM), Ibeurotoxin (IbTx; 100nM), or a vehicle control via mini‐osmotic pump. Complete gait analysis was performed using two‐dimensional motion capture by filming rats on a weekly basis for 8 weeks. Compound Action Potentials (CAP) and high frequency conduction experiments were performed on spinal cords 8 weeks post surgery. Finally, the size of the injury site, loss of myelination, and cell death were assessed using immunohistochemistry (IHC). Complete gait analysis suggests that treatment with ISO improves motor function in our test model. ISO treated animals were able to recover gait function to a greater extent than IbTx and control animals. While the IbTx treated rats also demonstrated an improvement in complete gait function, their recovery was delayed and not of the same magnitude as ISO treated animals. This finding was further supported in our electrophysiology and IHC experiments. In our CAP experiments we find that there is a significant difference between all three groups. Additionally, we found preservation or re‐establishment of axonal tracts and myelination in our IHC experiments. In conclusion, activation of the BK channel during acute SCI may be a novel therapeutic target for acute spinal cord injury. Further studies are required to determine if modulation of the BK channel in chronic injury is beneficial for recovery, and to understand the mechanisms by which this ion channel may be working during spinal cord injury repair.

Support or Funding Information

Saskatchewan Health Research Foundation
Human CLCA2 Modulates The Conductance Of Ano1 By Regulating Intracellular Calcium Level
Authors: Sharma Aarushi , Ramena Grace , Premkumar Louis , Elble Randolph C
Published: November 2023

Ano1 has recently been identified as a Calcium‐activated Chloride Channel (CaCC) that plays a key role in such physiological processes as nociception, signal transduction, smooth muscle contraction and secretion from airways and exocrine glands. Pathologically, Ano1 overexpression is associated with inflammatory disorders of airways. It is also amplified in certain cancers, including gastrointestinal, breast, and head and neck squamous cancers, in which it has been demonstrated to promote tumor proliferation. The Chloride Channel Accessory (CLCA) protein family was originally identified based on their ability to enhance an unknown CaCC by an unknown mechanism. Human CLCA2 is an epithelial junction molecule that is downregulated in most subtypes of breast adenocarcinoma but is upregulated along with Ano1 in certain breast cancer and head and neck squamous tumor subtypes. In view of this apparent functional link between the two proteins, we investigated whether human CLCA2 modulates the conductance of Ano1. Using whole‐cell voltage clamp electrophysiology, HEK293 cells co‐expressing heterologous CLCA2 and Ano1 exhibited larger chloride current amplitude in response to ionomycin‐mediated calcium influx than cells expressing either of the proteins alone. Protein cross‐linking and co‐immunoprecipitation studies did not detect any physical interaction between the proteins. In addition, surface biotinylation analysis revealed no increase in surface occupancy or multimerization of Ano1 in the presence of CLCA2. However, calcium imaging with Fluo‐4 AM revealed that CLCA2 increases both release of calcium from intracellular stores and subsequent activation of Store‐Operated Calcium Entry (SOCE) after treatment with calcium ionophore, ionomycin or a SERCA pump inhibitor, cyclopiazonic acid (CPA). In support of this finding, CLCA2 was shown to form a complex with ER calcium sensor STIM1 and SOCE channel ORAI1. The regulatory effects of CLCA2 were also observed with mutants having inactive metalloprotease or deleted intracellular domain, suggesting that the uncleaved ectodomain is responsible. Thus, CLCA2 modulates Ano1 conductance by enhancing both calcium loading of the ER and calcium influx via SOCE. Moreover, these results suggest that CLCA2 may play a broader role in calcium‐dependent processes than simply regulating CaCCs.
Nanomolar potency small‐molecule inhibitor of human ANO1/TMEM16A chloride channel
Authors: Namkung Wan
Published: November 2023

Anoctamin1 (ANO1)/transmembrane protein 16A (TMEM16A), a calcium‐activated chloride channel (CaCC), is involved in many physiological functions such as fluid secretion, smooth muscle contraction, nociception and cancer progression. To date, only a few ANO1 inhibitors have been described, and these have low potency and selectivity for ANO1. Here, we performed a high‐throughput screening to identify highly potent and specific small molecule inhibitors of ANO1. Three novel ANO1 inhibitors were discovered from screening of 54,400 synthetic small molecules, and they were found to fully block ANO1 channel activity with an IC50 < 3 μM. The most potent inhibitor, Ani9, completely inhibited ANO1 chloride current with IC50 approximately 77 nM, without interfering with intracellular calcium signaling. Notably, Ani9 was highly selective for ANO1 compared to ANO2, which shares a high amino acid homology to ANO1. In addition, Ani9 did not affect CFTR and ENaC channel activities, and compared to the previous potent ANO1 inhibitors such as T16Ainh‐A01 and MONNA, Ani9 had less effect on the VRAC activity. The first nanomolar human ANO1 inhibitor, Ani9, identified here permit pharmacological dissection of ANO1 and may be potential development candidates for drug therapy of cancer, hypertension, pain, diarrhea and asthma.
Proposed Mechanism for Post‐Exercise Weakness in Hypokalemic Periodic Paralysis
Authors: Cannon Steve C , Mi Wentao , Wu Fenfen
Published: November 2023

Hypokalemic periodic paralysis (HypoKPP) is a dominantly inherited channelopathy of skeletal muscle in which recurrent episodes of severe weakness are often provoked by environmental triggers such as carbohydrate ingestion, K+ loss, or exercise. Remarkably, the mutations that cause HypoKPP occur almost exclusively at arginine residues in the S4 voltage sensor domains of the L‐type Ca channel (CaV1.1) or the voltage‐gated sodium channel (NaV1.4). These R/X mutations of S4 produce an anomalous conduction pathway that is permissive (open) at hyperpolarized potentials. This so‐called gating pore leakage current renders the muscle fiber susceptible to anomalous depolarization of Vrest in low K+ (e.g. 2 to 3 mM). Depolarized fibers are refractory to the generation of action potentials, resulting in loss of force. While this low K+ induced basis for episodic weakness is well established, the mechanism by which weakness occurs within minutes of resting after strenuous exercise is completely unknown.

We tested the hypothesis that acidosis is a trigger for exercise‐induced attacks of weakness in HypoKPP. This notion was explored in our previously developed knock‐in mutant mouse models of HypoKPP (NaV1.4‐R669H and CaV1.1‐R528H). The soleus muscle was mounted in an HCO3 buffered tissue bath and maximal isometric force was measured during tetanic stimulation (100 Hz, 40 pulses). Acidosis was imposed by bath exchange with a solution equilibrated to 25% CO2 (pH 6.8), compared to baseline 5% (pH 7.4). Acidosis was well tolerated for WT and HypoKPP muscle during a 20 min exposure to 25% CO2. Upon return to 5% CO2, however, there was a marked transient decrease in force for HypoKPP muscle but not for WT. Myoplasmic pH homeostasis in response to the 25% CO2 challenge was indistinguishable for WT and HypoKPP muscle, as measured with BCECF. We propose the transient weakness is caused by a pH‐dependent rise of myoplasmic Cl. Acidosis reduces the ClC‐1 conductance (GCl), thereby limiting Cl efflux, while influx via the NKCC cotransporter produces a slow rise of myoplasmic Cl. A rapid correction of pH quickly increases GCl which depolarizes the fiber because of the shift of ECl. In support of this hypothesis, inhibition of NKCC with bumetanide completely prevents the post‐acidosis loss of force. Model simulations show myoplasmic Cl accumulation and susceptibility to depolarization are greater for HypoKPP muscle than WT due to the gating pore current.

We propose the post‐exercise weakness in HypoKPP muscle is caused by a shift of Cl into muscle while GCl is low during acidosis, followed by depolarization if GCl recovers quickly. A slower recovery from acidosis my allow the Cl gradient to return to baseline before a full recovery of GCl, thereby reducing the risk of paralysis as patients have reported with warm‐down after exercise.

Support or Funding Information

Supported by the National Institutes of Arthritis, Musculoskeletal, and Skin Diseases of the NIH (Grants AR42703 and AR063182).
Heparanase may contribute to idiopathic pulmonary fibrosis via nuclear translocation of cleaved heparan sulfate proteoglycans
Authors: Yang Yimu , Lara Abigail , Rutebemberwa Alleluiah , Schmidt Eric
Published: November 2023

Heparanase (HPSE) is an endo‐β‐glucuronidase capable of cleaving heparan sulfate (HS) side chains of heparin sulfate proteoglycans (HSPGs), important components of the extracellular matrix (ECM). HPSE has important roles in a variety of biological processes, including angiogenesis, inflammation, wound healing and metastasis. Little is known about HPSE and human idiopathic pulmonary fibrosis (IPF). Using immunohistochemistry, we noted that 6/6 human IPF lung biopsies expressed HPSE in vascular smooth muscle (VSM), endothelial cells and macrophages. In VSM and endothelium, heparanase was localized in both of the cytoplasm and nucleus. To determine if expressed HPSE was enzymatically active, we, immunostained with anti‐HS mAb 3G10 (which detects remnant HS fragment cleaved by HPSE). An abundance of 3G10 immunostaining were found in the nucleus of the most type of the cells. We hypothesized that prolonged HPSE activity cleaves HS in the ECM/cell surface, inducing an internalization and nuclear translocation of HSPGs. These findings were confirmed using mouse lung fibroblasts treated with heparinase‐III, a HPSE analog, which demonstrated nuclear translocation of 3G10 positive (HS‐fragmented) proteoglycans. Our findings suggest that nuclear HS/HSPGs may be directly involved in regulating biological processes including transcription and cell cycle progression, leading to the abnormal cell proliferation and wound repair characteristic of IPF.

Support or Funding Information

K08 HL105538
Does Rho Inhibition Alter Microtubule Acetylation and Detyrosination?
Authors: McGee James P , Danowski Barbara A
Published: November 2023

Microtubules (MTs) play a role in many cellular functions, such as chromosome segregation, establishment of polarity, and vesicle transport. Numerous post‐translational modifications (PTMs) to tubulin have been identified, but their functions are still not well‐understood. Microtubules are also known to modulate actin‐based cell contractility; changes in MT dynamics have been shown to play a role in this crosstalk. Actin‐myosin contractility is mediated through rho, a member of the Rho family of GTPases. Prior studies have shown a possible relationship between increased contractility due to rho activation and specific tubulin post‐translational modifications. In order to investigate this further, we examined whether the amount and/or localization of acetylated and detyrosinated MTs changes when rho is inhibited. Immunofluorescence was performed on C3H10T1/2 mouse embryo fibroblasts that were treated with a membrane‐permeant rho inhibitor for 1 & 4 hours and evaluated for alpha‐tubulin and either detyrosinated or acetylated tubulin. Cells were scored based on the presence of high, medium or low amounts of each of the post‐translational modifications. Based on 3 separate experiments, we found a statistically significant decrease in the amount of acetylated MTs but no difference in detyrosinated MTs. Western blotting will be done to determine whether rho inhibition alters the concentrations of the modified tubulins in cell extracts. These results could shed further light on the mechanisms of microtubule‐actin cytoskeletal crosstalk and provide insight into the role of acetylated microtubules.
MED28 regulates TNFα‐ and CoCl2‐mediated migration and invasion in human non‐small cell lung cancer (NSCLC) cells
Authors: Hsieh Nien‐Tsu , Huang Chun‐Yin , Cheng An‐Chin , Lee Ming‐Fen
Published: November 2023

Growing evidence indicates that chronic inflammation and oxidative stress are involved in the malignant progression. Epithelial tumors undergo epithelial‐mesenchymal transition (EMT) and remodel extracellular matrix to acquire a motile and invasive phenotype. MED28, a Mediator subunit for transcriptional activation, is also found highly expressed in several types of epithelial tumors. Our laboratory has recently reported that MED28 upregulates cell growth in both human breast cancer and colorectal cancer cells, and MED28 modulates migration and invasion in MCF7 and MDA‐MB‐231 human breast cancer cells. The present study was to explore the potential role of MED28 in inflammation‐ and oxidative stress‐induced EMT and migration in A549 human non‐small cell lung cancer (NSCLC) cells. Cellular oxidative stress was increased upon the addition of either TNFα or CoCl2. Both TNFα and CoCl2 induced EMT and enhanced migration as evidenced by increasing mesenchymal cell types and Matrigel invasion, respectively, and accompanied by concomitantly upregulating the target genes and the functional activity of NFκB. However, these upregulation events were attenuated upon MED28 knockdown. Out data indicate that MED28 modulates oxidative stress, EMT, and invasion in human NSCLC cells, further strengthening a critical role of MED28 in cancer development.

Support or Funding Information

This work was supported by the grant NSC102‐2320‐B‐309‐001‐MY3 to M‐F Lee.
Effects of calreticulin on FUT1 expression and beta1 integrin fucosylation in different cancer cells
Authors: Weng Yi‐Cheng , Chien Yin‐Chieh , Lu Kuan‐Ying , Lee Hsinyu
Published: November 2023

Calreticulin (CRT) is a multifunctional ER chaperon protein. We had demonstrated that knockdown of CRT may suppress cell proliferation and migration in J82 bladder cancer cells. We further revealed that CRT stabilized the mRNA of fucosyltransferase1 (FUT1), which leads to an enhancement of fucosylation and the subsequent activation of beta1 integrin. Previous studies had demonstrated that knockdown of CRT suppress cell proliferation and migration in both PC3 prostate cancer cells and AGS gastric cancer cells. However, the effects of CRT on FUT1 expression and beta1 integrin fucosylation have not been investigated in these cells.

In this study, we demonstrated that knockdown of CRT decreased the expression levels of FUT1 in PC3 cells and AGS cells. Furthermore, the fucosylation levels of beta1 integrin were significantly down regulated in these CRT knockdown cells. However, mRNA stability of FUT1 was not affected in either cell lines. These results suggested that CRT might affect the fucosylation of beta1 integrin in both PC3 cells and AGS cells. However, a different molecular mechanism was involved while compared to J82 bladder cancer cells. Taken together, our observations imply that CRT may regulate FUT1 expression differently between different cancer cells.