bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of TRPV4‐stimulated Ion Transport in Choroid Plexus Epithelia by Arachidonic Acid
Authors: Otun Ayodamola , Preston Daniel , Danko Caleb , Schroten Horst , Schwerk Christian , Blazer‐Yost Bonnie 
Published: November 2023

Hydrocephalus is a condition that can be caused by overproduction of cerebrospinal fluid (CSF) in the choroid plexus (CP) seated in the brain ventricles. Our laboratory has recently discovered that an antagonist of an osmotic‐ and mechano‐sensitive calcium channel, TRPV4, is effective in treating hydrocephalus in a rat model. Hydrocephalus involves a high level of inflammation due to the fluid pressure exerted on brain tissue. It is, therefore, of interest to study the role of inflammation on ion transport mediated by TRPV4 so as to further understand endogenous modulators involved in hydrocephalic development. To determine the effect of potential endogenous mediators, we conducted Ussing style electrophysiological experiments to measure transepithelial ion transport in a high resistance porcine choroid plexus cell line (PCP‐R). This cell line has been shown to demonstrate similar characteristics as the native choroid plexus and responds to the TRPV4 agonist GSK1016790A with an increase in transepithelial ion flux and a decrease in transepithelial resistance. Arachidonic acid is an endogenous molecule produced during an inflammatory response and is also a precursor to multiple prostanoids some of which are known to activate TRPV4. A short (10 minute) pre‐incubation with arachidonic acid has no effect on basal ion flux in confluent PCP‐R cells but potentiates TRPV4‐stimulated transepithelial ion flux by approximately 150%. The transepithelial resistance of the cells was not altered in response to arachidonic acid but arachidonic acid did not alter the TRPV4‐mediated decrease in resistance. It is hypothesized that the overproduction of fluid in hydrocephalus is due, in part, to the abnormal flux of ions into the CSF and, likewise, inflammatory responses cause changes in ion and fluid balance. These data indicate that either arachidonic acid or a metabolite formed by enzymes in the choroid plexus cells modulate ion transport responses rather than initiate them. These studies are important for understanding the formation of excess CSF during hydrocephalic development and in other inflammatory brain diseases.Support or Funding InformationHydrocephalus Association Innovator Award; Indiana University Collaborative Research Grant and Indiana Clinical and Translational Sciences Institute CTR Award.
Activation of TRPA1 and TRPV1 in vagal nociceptive sensory neurons by inhibitors of mitochondrial function
Authors: Stanford Katherine R , Barannikov Ivan R , Taylor‐Clark Tom E 
Published: November 2023

Inflammatory signaling pathways such as tumor necrosis factor alpha and interleukin‐1β result in mitochondrial dysfunction and induce mitochondrial reactive oxygen species (ROS) production, depolarization, calcium release as well as morphological changes such as remodeling and fission. Mitochondrial dysfunction is linked to nociceptive disorders which involve sensory nerve hyperexcitability including asthma, and diabetic painful neuropathy. However, it is not known how mitochondrial signaling events may influence nociceptive signaling. Our lab has previously demonstrated that Antimycin A, a mitochondrial electron transport chain complex III inhibitor, activates bronchopulmonary C‐fibers via gating of transient receptor potential channels (TRP) Ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). Both TRPA1 and TRPV1 are selectively expressed in nociceptive subtypes of vagal, trigeminal and dorsal root afferents. Here we characterized the response to three inhibitors of mitochondrial function: Rotenone (complex I inhibitor), Carbonyl cyanide m‐chlorophenyl hydrazine (CCCP, mitochondrial inner membrane proton ionophore), and Antimycin A (complex III inhibitor) using live cell imaging of dissociated vagal neurons. We used co‐imaging of mitochondrial superoxide (MitoSox Red) and calcium (FURA‐2AM) to simultaneously measure ROS production and neuronal activation. Antimycin A and CCCP caused ROS production in both nociceptive and non‐nociceptive neurons. Rotenone had little effect on ROS production. Ca2+ imaging showed that mitochondrial modulators failed to activate non‐nociceptive neurons, but selectively stimulated approximately 50% of nociceptive neurons. Interestingly, this activation was not correlated with ROS production. Antimycin A‐induced nociceptor activation was abolished in TRPA1−/− neurons pretreated with the TRPV1 inhibitor iodoresiniferatoxin. However, only a fraction of the Antimycin A‐induced nociceptor activation was prevented by the reducing agent dithiothreitol (DTT). Taken together, this suggests that modulation of mitochondrial function activates nociceptive neurons via ROS‐mediated and ROS‐independent mechanisms linked to TRPA1 and TRPV1.Support or Funding InformationNHLBI R01 (HL119802)
Selection of High Affinity Binders Against the Thumb‐Domain of αENaC using a Bacterial Two‐Hybrid System
Authors: Svenningsen Per , Dimke Henrik 
Published: November 2023

The objective of project was to develop reagents that facilitate the isolation of extracellular vesicles (EVs) containing the epithelial Na+ channel (ENaC). The ENaC subunits are highly conserved, and the traditional method of generating antibodies against the extracellular domain of ENaC does often not produce high affinity binders. We therefore sought to develop a recombinant affinity reagent using a bacterial two‐hybrid screen. The thumb domain of αENaC (Gly415‐Lys504) was used as bait to isolate high affinity binders. As binders, we generated a library of the small antibody mimetics termed affibodies. The isolated affibodies was subsequently characterized by producing a fusion protein consisting of biotin‐acceptor peptide (AP), the affibody and an intein/chitin‐binding domain. The affibody fusion protein was readily expressed in bacteria and was purified using chitin‐resin. Subsequently, the AP‐fused affibody was effectively eluted from the chitin beads using thiol‐induced cleavage. As demonstrated by streptavidin gel‐shift assay, co‐expression of the AP‐fusion protein with biotin protein ligase BirA biotinylated the affibody, thereby producing an effective tag for the subsequent isolation of the affibody. One of the affibodies was further tested in preliminary experiments for its ability to isolate ENaC positive urinary EVs. Compared to urinary EVs isolated by ultracentrifugation, affinity isolated samples were depleted in Tamm‐Horsfall protein as judged by coomassie‐stained SDS‐PAGE. Immunoblots showed that urinary EVs isolated by affibodies and ultracentrifugation were positive for αENaC, AQP2, and the exosomal marker ALIX. Thus, our results indicate that affibodies are useful affinity reagents for enrichment of specific urinary EVs.Support or Funding InformationLundbeck Foundation and Novo Nordisk Foundation
Intracellular and Near‐Membrane Dynamics of L‐type CaV1.2 Channel
Authors: Ghosh Debapriya , Nieves‐Cintrón Madeline , Navedo Manuel F 
Published: November 2023

L‐type CaV1.2 channels (LTCCs) are involved in myriad of physiological functions ranging from excitation, contraction, transcription and proliferation. Numerous studies had been conducted to understand the electrophysiological and biochemical properties of LTCC. Yet, the intracellular and near‐membrane dynamics of LTCCs are poorly understood. Here, using 4D spinning‐disk‐confocal and near‐membrane Total Internal Reflection Microscopy (TIRFM) recordings, we found that LTCCs exhibit diverse and dynamic intracellular trafficking patterns executed via compartments in form of punctate and larger ovoid vesicles as well as elongated tubules. Total Internal Reflection Fluorescence Recovery After Photobleaching (TIR‐FRAP) showed that both actin and microtubules are equally important in active plasma membrane trafficking of these compartments, where LTCC demonstrate either ‘kiss and run’ or ‘kiss and linger’ type fusion events, while perhaps also supporting distinct patterns of intracellular LTCC movements. These LTCC carrying compartments seems to undergo constitutive homotypic fusion and fission events, near the cell membrane. In conclusion, our study reveals very diverse and unique intracellular and near‐membrane dynamics of LTCCs with profound implications for ion channel trafficking.Support or Funding InformationThis work is supported by NIH R01HL098200 and R01HL121059.
Cardioprotection by Genetic Deletion of Kvβ2 in Ischemia Reperfusion Injury
Authors: Chapalamadugu Kalyan C. , Badole Sachin L. , Tur Jared , Tipparaju Srinivas M. 
Published: November 2023

BackgroundIschemia reperfusion (I/R) injury increases the levels of NADH in the heart leading to increased NADH/NAD+ ratio. Voltage‐gated potassium channel (Kv) β‐subunits bind to ion channels and contractile proteins resulting in modulation of cardiac function. Previous studies demonstrated high affinity binding of voltage‐gated potassium channel beta subunit 2 (Kvβ2) to pyridine nucleotides (NAD[P]+, NAD[P]H).HypothesisWe hypothesized that Kvβ2 plays a major role in sensing I/R injury.Methods and ResultsHearts from wild type (WT) and Kvβ2 knockout (KO) mice were perfused using Langendorff apparatus and subjected to 45 min no‐flow global ischemia followed by 120 min reperfusion. At the end of reperfusion, hearts were sectioned and stained with 2,3,5‐triphenyltetrazolium chloride (TTC). Cardiac perfusates were collected before ischemia and during reperfusion at 0, 30 and 120 min, and cardiac troponin I (cTnI) was measured. Intracardiac levels of NAD+ and NADH were measured in I/R injured hearts. Infarct size was measured, and normalized to total area of the heart. I/R challenge of WT mice hearts resulted in a significant myocardial damage where the infarct size was 46.6±5.4%. However, infarct size in Kvβ2 KO hearts was only 12±2.3% (n=5), which clearly demonstrates that the KO hearts show significant resistance to injury (p<0.05), despite similar changes in NADH/NAD ratio in both groups. Baseline cTnI levels (ng/ml) in the perfusate were similar between the WT and KO hearts (n=3). During reperfusion, cTnI levels in the perfusate from KO hearts were significantly (p<0.05) decreased when compared to WT.ConclusionsThis study shows that Kvβ2 deletion is cardioprotective in I/R injury. It is plausible that Kvβ2:NADH/NAD axis may serve as a metabolic sensor in I/R injury, where Kvβ2 subunit senses and relays the increased NADH/NAD levels to target proteins in WT hearts, whereas deletion of Kvβ2 abrogates relaying the information leading to protection.
ClC‐3 and LRRC8A interact physically and co‐localize with endosomal superoxide production.
Authors: Nguyen Hong‐Ngan , Choi Hyehun , Lamb Fred S 
Published: November 2023

Both the ClC‐3 Cl−/H+ antiporter and LRRC8A anion channels are required for TNFα‐induced, Nox1‐dependent superoxide (O2−•) production and subsequent signaling in vascular smooth muscle cells (VSMC). Both proteins have been associated with the swelling‐activated anion conductance (IClswell) which can modulate Nox1 activity, and thereby TNFα signaling. It is now clear that LRRC8 family (LRRC8A‐E) proteins constitute IClswell channels. However, it is not known why ClC‐3 expression also affects the magnitude of this anion current. We hypothesized that a physical relationship and functional interdependence between ClC‐3 and LRRC8A explains this finding. Confocal microscopy and immunoprecipitation were used to localize expressed ClC‐3 and LRRC8A with O2−• (ROSstar 550) in HEK293 cells expressing fluorescent fusion proteins. The endogenous proteins were localized by immunostaining of cultured human aortic VSMC. As previously demonstrated, ClC‐3‐GFP localizes to the membrane of very large intracellular vesicles (1–10 μm diameter). These compartments are now revealed to be multivesicular bodies, and the smaller vesicles within (0.1–0.6 μm) are a prominent site of ROSstar staining. When expressed alone, GFP‐LRRC8A localizes to small cytoplasmic vesicles which also are strongly ROSstar positive, suggesting that this is an important site of O2−• production. Co‐expression of ClC‐3‐GFP and mCherry‐LRRC8A reveals that small vesicles, which are rich in LRRC8A, are contained within ClC‐3‐expressing multivesicular bodies. In VSMC, antibodies to ClC‐3, LRRC8A and Nox1 stain both localized regions of the plasma membrane and scattered intracellular vesicles in the 0.1–0.60 μm range. Overlapping staining for all three proteins is seen in a subset of these vesicles, and in scattered multivesicular clusters. We have previously demonstrated that LRRC8A co‐immunoprecipitates with Nox1 and its p22phox subunit (Choi, FRBM, 2016). We now observe that immunoprecipitation of endogenous ClC‐3 pulls down LRRC8A. These findings demonstrate co‐localization and physical association between ClC‐3 and LRRC8A. This association, at sites where Nox1 is producing O2−•, may explain why both proteins play a role in Nox1‐mediated O2−• production and TNFα signaling.Support or Funding InformationHC is supported by an SDG from the American Heart Association. FL is supported by R01 HL128386.
Cardiac structure and function in mice with a cardiomyocyte‐specific knockout of Na+/K+‐ATPase α1‐isoform
Authors: Marck Pauline V , Wang Xiaoliang , Kutz Laura , Cui Xiaoyu , Blanco Gustavo , Cai Liquan , Xie Zijian , Pierre Sandrine V 
Published: November 2023

Owing mostly to the clear impact of the Na/K‐ATPase specific ligand ouabain and other cardiac glycosides (CG) on cardiac contractility and myocyte size, the importance of both Na/K‐ATPase ion‐pumping and signaling functions in the regulation of cardiac structure and performance have been recognized for decades. In the mouse heart, about 80% of Na/K‐ATPase activity is carried on by α1‐containing enzyme complexes relatively insensitive to ouabain, and the remainder is supported by ouabain‐sensitive α2‐containing enzymes. Evidence obtained in mammalian cell models expressing either one of these isoforms suggest that they serve highly specific cellular functions, but it has been technically challenging to investigate them in a cardiac myocyte and assess the impact on cardiac structure and function. We generated a mouse model with a cardiomyocyte‐selective Na/K‐ATPase α1‐isoform knockout using α‐myosin heavy chain (Myh6) cre/loxP recombination. Animals with a cardiac α1−/− genotype (cardiac α1−/−) were born with the expected Mendelian frequency and survived to adulthood. Western blot analysis confirmed the absence of detectable α1 protein with an apparent compensatory 2.6 fold upregulation of α2 expression without altering Na/K‐ATPase β1 isoform expression in cardiac α1−/− hearts. No major changes in heart weight/body weight or heart weight/tibia length ratios were noted in a cohort of males at 4 month of age. Transthoracic echocardiography studies in isoflurane‐anesthetized animals did not reveal a substantial adverse impact on cardiac function in this cohort, and a mild but significant increased stroke volume was noted in cardiac α1−/− mice (+27%, 23 μl± 1 μl vs 29 μl± 1 μl, P<0.01, n=5–6). These results suggest that the cardiac α1−/− is viable and shall provide a useful model in which to study the role of Na/K‐ATPase α‐isoforms and the impact of cardiac Na/K‐ATPase signaling in health and diseases.Support or Funding InformationMarshall Institute for Interdisciplinary Research and NIH Grant HL‐109015
Na/K‐ATPase α1 Isoform as a Critical Signal Integrator in Embryonic Development
Authors: Wang Xiaoliang , Zhang Jue , Cui Xiaoyu , Wang Jiayan , Cai Liquan , Pierre Sandrine V , Xie Zijian 
Published: November 2023

We have shown that Na/K‐ATPase interacts with caveolin‐1 through a putative caveolin‐binding motif, and regulates its trafficking in cell culture. The aim of this study is to review the importance of this putative interaction in animal physiology. A mCBM (F97A and F100A) mutant mouse was generated using a knock‐in strategy. Homozygosity of the mCBM Na/K‐ATPase animal results in embryonic lethality around mid‐gestation. Heterozygous animals were born with the expected Mendelian frequency and survived to adulthood. Further analysis of the embryonic development reveals the following defects. The first phenotype of homozygous embryos with obviously defect was noted at E7.0 day. At E9.0 day, homozygous embryos exhibited severe growth retardation with defective brain and somite development. By E12.5 day, the homozygous embryos were no longer detectable. On the other hand, heterozygous embryos seems to be normal through the development. However, they are significantly smaller in size at E9.0 day. Mechanically, RNAseq shows normal expression of different isoforms of Na/K‐ATPase and caveolin‐1. However, it reveals defects in Wnting signaling pathway, which results in severe down‐regulation of genes important for neurogenesis and muscle genesis. Those defects were further confirmed by qPCR analysis of mRNA from wild type and homozygous embryos. Taken together, our new findings suggest that α1 Na/K‐ATPase functions as a critical signal integrator in animal embryonic development, and that the putative caveolin‐binding motif is required for this non‐pumping function of Na/K‐ATPase.Support or Funding InformationMarshall Institute for Interdisciplinary Research and NIH Grant HL‐109015
Uptake and Protein Interaction of APOL1 with HSP60 in the Proximal Tubule
Authors: Blessing Natalya A , Konieczkowski Martha , Madhavan Sethu , O'Toole John , Sedor John , Bruggeman Leslie 
Published: November 2023

Coding variants of APOL1, common in African Americans, have been linked to the elevated risks of chronic kidney disease (CKD) associated with African ancestry. While all variants of apolipoprotein 1 (APOL1) circulate in the serum bound to HDLs and confer protection against Trypanosoma brucei brucei, APOL1 G1 and G2 variants offer additional protection against T.b. rhodesiense. T.b. rhodesiense produces serum‐resistance associated (SRA) protein, which binds to the C‐terminal region of APOL1 G0 and blocks trypanolysis. APOL1 G1 and G2 evade the trypanolysis block through a reduced (G1) or nonexistent (G2) interaction with SRA. Preliminary studies have demonstrated that both APOL1 protein and mRNA are present in glomerular podocytes; however, mRNA in situ hybridization failed to detect APOL1 transcripts in the proximal tubule (PT), despite APOL1 protein being most abundant in the mitochondria of PT cells. APOL1 function in the context of kidney disease and the route of apparent uptake into the PT remains unknown. To investigate the renal function of APOL1, we hypothesized that APOL1 variant‐dependent interaction with SRA implies a variant‐dependent interaction between APOL1 and an SRA‐like binding partner in humans that contributes to APOL1‐associated CKD. To identify human APOL1‐interacting proteins, a yeast‐two‐hybrid screen of human cDNA library using the SRA‐interacting domain of APOL1 as bait detected heat shock protein 60 (HSP60), a stress response chaperone for mitochondrial proteins, as a binding partner for APOL1. Confirmatory yeast two hybrid and immunoprecipitation experiments observed differential binding of APOL1‐G0 compared to the CKD variants, G1 and G2. With the HSP60 connection to the mitochondria, we hypothesize that APOL1 is imported into the proximal tubule from the basal (serum) and is trafficked to the mitochondria. As a result of APOL1‐associated CKD stress, imported APOL1 loses proper conformation and requires HSP60 for refolding. APOL1 G1 and G2 variants lack the appropriate residues in the HSP60 binding site, resulting in a misfolded conformation and improper signaling. To test the uptake of APOL1 in vitro, HK‐2 cells, which do not express APOL1, were cultured with 10% human serum for a timecourse extending up to two days, harvested and immunoblotted with APOL1 antibody. APOL1 protein levels, but not mRNA levels, were detected after 24 hours of treatment, suggesting an uptake of serum APOL1. The preliminary findings support the hypothesis of APOL1 entering through proximal tubule from the serum and having a possible functional role in the mitochondria involving HSP60.Support or Funding InformationNIH DK0007470
The Ca2+‐Activated Chloride Channel TMEM16A Colocalizes with CFTR Chloride Channels in Rodent Secretory Airway Epithelial Cells
Authors: Hahn Anne K , Faulhaber Johannes , Srisawang Lalita , Salomon Johanna J , Mall Marcus M , Möhrlen Frank , Frings Stephan 
Published: November 2023

There is a variety of animal models available to study cystic fibrosis, a disease caused by mutations in the CFTR gene. However, the applicability of these models is limited as CFTR−/− mice and also CFTR deficient rats fail to recapitulate phenotypic features of the human disease in the lung. One reason may be compensatory effects of Ca2+‐mediated Cl− secretion in airway epithelia. The purpose of this study was to investigate whether the chloride channel TMEM16A accounts for the molecular basis of Ca2+‐activated Cl− conductance in rodent airway epithelia. Furthermore, the spatial expression of the chloride channels, TMEM16A and CFTR, was studied in tissue of the respiratory system of rats and mice.Rat tracheal epithelial cells were cultured under air‐liquid culture conditions for at least 14 days and subsequently the short circuit current (ISC) was measured in Ussing chambers. For immunohistochemical stainings, respiratory epithelia of the nose, trachea and lungs of mice and rats were co‐stained using anti‐TMEM16A and anti‐CFTR antibodies in combination with markers for different epithelial cell types (e.g. ciliated cells, goblet cells, club cells)Bioelectrical studies revealed that the UTP‐induced increase in ISC reflecting the Ca2+‐activated Cl− secretion was significantly inhibited by 95% after preincubation with the specific TMEM16A inhibitor Ani9 (ISC values of 0,6 ± 0,5 μA/cm2 (pretreated) vs.12,6 ± 4,3 μA/cm2 (non‐treated)) in rat tracheal epithelial cells. In contrast to human airway epithelia, immunohistochemical stainings in rodents showed a colocalization of TMEM16A and CFTR in secretory epithelial cells throughout the respiratory system. Both channels were not expressed in ciliated cells.In conclusion, TMEM16A inhibitor Ani9 selectively inhibits UTP‐induced Ca2+‐activated Cl−‐ currents accounting for TMEM16A‐mediated Cl− conductance. The colocalization of TMEM16A and CFTR in secretory cells of rodents suggests a potential regulatory interaction.Support or Funding InformationStudienstiftung des deutschen Volkes