bluuebunny/crossref_metadata_2025 huggingface dataset:
N‐Terminal α2 Na+ Pump Constructs are Dominant Negative for Native α2 Na+ Pumps in Smooth, but not Cardiac Muscles, and Increase Salt Sensitivity
Authors: Song Hong , Chen Ling , Lee Jane C , Kotlikoff Michael I , Lederer W Jonathan , Frederick Cecilia A , Karaashima Eiji , Randall Willaim C , Blaustein Mordecai P
Published: June 2021

Plasmids that encode the N‐terminal 90–150 AAs of Na+ pumps with an α2 catalytic subunit are dominant negative (DN) for α2 expression in astrocytes and artery myocytes (Song et al., J Biol Chem 281:12929, 2006). To test the effect of reduced myocyte α2 on mean blood pressure (MBP), we generated α2SM/DN mice that express α2(1‐120)/flag under smooth muscle (SM) myosin heavy chain promoter control. Normal α2 Na+ pumps as well as Na/Ca exchanger‐1 (NCX1) were markedly downregulated in artery and urinary bladder SMs, but not brain. MBPs were recorded by telemetry during 12 hr light (low locomotor activity) periods of 12 hr dark/light cycles. Baseline MBP (0.5% salt diet) did not differ in α2SM/DN mice and wild type (WT) siblings (99 ± 2 vs 97 ± 2 mm Hg; n = 6 each), perhaps because of low NCX1 expression. Nevertheless, MBP increased in α2SM/DN (106 ± 4 vs baseline; P <0.03, ANOVA), but not WT (98 ± 3 vs baseline; NS), mice fed a 6% salt diet + 1% NaCl drinking water. Thus, reduced SM α2 expression increases salt sensitivity. Rat cardiomyocytes, which express α2 Na+ pumps, were transfected (viruses) with mCherry/α2(1‐150)/flag120 or α2(1‐130)/α1‐flag chimera. Cardiomyocytes expressed both constructs (in confocal, mCherry expressed in transverse and longitudinal tubules), but neither downregulated α2. Thus, unlike SMs and astrocytes, the cardiomyocyte α2 N‐terminus may be necessary, but is insufficient to displace normal α2 Na+ pumps.
Sorting of α2 and α3 Na+ Pumps in Glia and Neurons: Linkage with Na/Ca Exchanger‐1
Authors: Song Hong , Weber David J , Thompson Scott M , Blaustein Mordecai P.
Published: June 2021

The α2 Na+ pump amino acid (AA) sorting sequence in astrocytes and arterial myocytes is: L27DELKKEVA35 (Song et al., J Biol Chem 281:12929, 2006). Plasmids encoding this sequence, with an N‐terminal Green Fluorescent Protein and C‐terminal Flag (f)‐tag (GFP‐LDELKKEVA‐f), when transfected (Lipofectamine) into mouse primary cultured astrocytes, were dominant negative (DN) for normal α2 expression (by immunocytochemistry). Mutations of AAs 28–34 to Ala did not prevent the DN effect, but L27A, A35S (as in α1 pumps) or K31P were not DN. Thus, the α2 sorting sequence corresponds to two turns of an α‐helix with attachment sites at Leu‐27 and Ala‐35. Loss of the DN effect by the K31P mutant is consistent because Pro disrupts α‐helices. Adenoviral transfection of α2(1‐130)/α1‐f chimera in astrocytes knocks down α2 Na+ pumps and, simultaneously, Na/Ca exchanger 1 (NCX1) expression, with a ~12 hr half‐time. NCX1 silencer RNA and α2 Na+ pump knock‐out mouse data also reveal linkage of α2 and NCX1 expression in astrocytes.

The homologous neuronal α3 Na+ pump N‐terminal sequence is: LDDLKKEVA. We generated plasmids coding for the N‐terminal 120 AAs of α1 and α3 Na+ pumps with mCherry and flag tags [mCherry‐α1(1‐120)‐f and mCherry‐α3(1‐120)‐f]. Transfection and expression of the latter, but not the former, was DN for α3 Na+ pumps in rat primary cultured hippocampal neurons. This suggests that LDDLKKEVA is the α3 sorting sequence.
POSSIBLE ROLE OF ADAPTOR PROTEINS IN THE TRAFFICKING OF hIK1
Authors: McKenzie Jose , Hamilton Kirk L.
Published: June 2021

We are now beginning to understand the trafficking of the intermediate‐conductance Ca2+‐activated K+ channel (hIK1). hIK1 possesses a di‐leucine (L18/L19) motif in the N‐terminus, however, when this motif is mutated (L18A/L19A), the channel fails to traffick properly. In this study, we examined the role this di‐leucine motif plays in the trafficking of hIK1. HA‐tagged channel constructs used included wild‐type HA‐hIK1, HA‐hIK1‐L18A, HA‐hIK1‐L19A and HA‐hIK1‐L18A/19A stably expressed in HEK cells. We demonstrated, by Western blot, that HA‐hIK1‐L18A and HA‐hIK1‐L19A were not expressed at the level of the wild‐type channel, however, HA‐hIK1‐L18A/19A did not appear to be expressed. To determine whether these channels were being rapidly degraded by lysosomes, we cultured wild‐type HA‐hIK1 and the mutated channels with the lysosomal pathway inhibitors leupeptin (L,15 μM) and pepstatin (P, 15 μM) for 16 hrs. L/P did not enhance the amount of protein of any of the channel constructs suggesting that hIK1 and the mutated channels were not degraded by the lysosomal pathway. Next, we incubated channels with MG132 (20 μM, 16 hrs), a proteosome inhibitor, to determine if these channels were degraded by proteasomes. Indeed, there was an increase in total protein of all channels in the presence of MG132. Finally, we used brefeldin A (BFA, 10 μg/ml, 4 hrs) to establish whether adaptor protein 1 (AP‐1) was involved in the trafficking of hIK1. BFA resulted in an increase in protein levels of all of the channels. Our results suggest that hIK1 and the mutated channels were trafficked in an AP‐1‐dependent mechanism and that the channels were degraded by the proteasome. This study was funded by the Department of Physiology at the University of Otago.
Golgi Trafficking Defect in Channelopathy Reveals a New Cargo Selection Mechanism
Authors: Taneja Tarvinder Kaur , Ma Donghui , Hagen Brian M , Kim Bo‐Young , Lederer Jonathan , Welling Paul A
Published: June 2021

Newly synthesized proteins destined for the plasmalemma are thought to be excluded from clathrin‐coated vesicles at the Golgi, and simply sorted to the cell surface by default. While exploring the basis of a human disease (Andersen‐Tawil Syndrome) mutation in the Kir 2.1 potassium channel, which we found surprisingly arrests traffic at the Golgi, we discovered this is not the case. Instead, channels are selected as cargo into Golgi export carriers through a unique signal‐dependent mechanism. Unlike typical trafficking signals, which are comprised of short linear sequences, the Golgi exit signal in Kir 2.1 is embedded within the confluence of two separate cytoplasmic domains. Forming a novel recognition site for interaction with the AP1 adaptin complex, the structure effectively marks Kir2.1 as cargo for incorporation into clathrin‐coated vesicles at the trans‐Golgi. Indeed, siRNA‐mediated AP‐1 knockdown prevented Golgi‐export, leading to the accumulation of the channel at the Golgi, just like channels bearing mutations in the Golgi export signal. The unique location of the trafficking signal in the tertiary structure implies that it functions in a previously unrecognized quality control step, coupling protein conformation to Golgi export.
Gating‐associated conformational changes in the pore of ASIC1a
Authors: Tolino Lindsey A , Okumura Sora , Carattino Marcelo D
Published: June 2021

Acid‐sensing ion channels (ASICs) are trimeric cation channels that undergo concerted activation and desensitization in response to extracellular acidification. Each subunit has two transmembrane domains (TMs) connected by a large extracellular region with intracellular N‐ and C‐ terminus. The mechanism of gating and desensitization of ASICs remains undefined. We performed studies of accessibility to covalent modification by MTSET of residues in the pore of ASIC1a in the closed and desensitized states. The activity of channels carrying cysteine mutations in the TM1 (tract V65‐F69) and TM2 (tract I426‐G435) was analyzed with the two‐electrode voltage clamp technique. The response to extracellular acidification after MTSET treatment was significantly affected in Y67C, I426C, A427C and G428C channels. The accessibility to covalent modification of cysteine residues at position 67, 426 and 427 was similar in the closed and desensitized states. Positively charged thiol‐modifying reagents (MTSEA and MTSET) activate G428C channels in the desensitized state, but only partially in the closed state. MTSES, which is negatively charged, does not activate G428C channels, but reduces the relative response to extracellular acidification. Our results indicate that the MTSET‐modified side chain of cysteine at position 428 interacts with a neighbor residue inducing a conformational change that opens the pore.
C‐Terminal threonines in the KCC4 K‐Cl cotransporter confer sensitivity to cell volume
Authors: Mercado Adriana , Babilonia Elisa , Gagnon Kenneth B. , Mandal Asim , Delpire Eric , Mount David B.
Published: June 2021

Cation‐chloride cotransporters exhibit reciprocal responses to cell volume, with activation of K‐Cl cotransporters (KCCs) under hypotonic conditions and of Na‐K‐2Cl cotransporters (NKCC1/2) under hypertonic conditions. N‐Terminal phosphorylation by the WNK‐activated SPAK/OSR1 kinases activates NKCCs; in contrast, the KCCs are inhibited by co‐expressed WNK4/SPAK. To identify the residues responsible for volume sensitivity we have systematically screened KCC4 by mutagenesis of serine/threonine residues, followed by assessment of transport activity in Xenopus oocytes under isotonic and hypotonic conditions, +/− WNK4/SPAK. A compound mutant of all 17 N‐terminal serine and threonines has no activity under isotonic conditions, with preserved swelling‐activated transport and inhibition by WNK4/SPAK. Screening of C‐terminal serines and threonines identified several that confer resistance to WNK4/SPAK; alanine mutants of three C‐terminal threonines are constitutively active under isotonic conditions, with analogous effects in KCC2. However, C‐terminal KCC4 fusion protein is not an in vitro substrate for SPAK fusion protein, which phosphorylates N‐terminal NKCC1 fusion protein. In summary, we have identified three C‐terminal threonines that confer volume and WNK/SPAK sensitivity to KCC4; these residues do not however appear to be direct substrates for the SPAK serine‐threonine kinase.
Na+/H+‐EXCHANGER‐1 INHIBITION REVERSES PERIPHERAL DIABETIC NEUROPATHY
Authors: Lupachyk Sergey , Watcho Pierre , Maksimchyk Yury , Shevalye Hanna , Oltman Christine , Yorek Mark A , Obrosova Irina G
Published: June 2021

Evidence for the important role of Na+,H+‐exchanger‐1 (NHE‐1) in diabetic complications is emerging. The aim of this study was to evaluate if NHE‐1 inhibition reverses experimental peripheral diabetic neuropathy (PDN). Control and streptozotocin (STZ)‐diabetic rats were maintained with or without treatment with the specific NHE‐1 inhibitor cariporide for 4 weeks after first 12 weeks without treatment. Rats with 12‐week duration of STZ‐diabetes developed motor and sensory nerve conduction slowing, neurovascular dysfunction manifest by deficit in endoneurial nutritive blood flow and impaired vascular reactivity of epineurial arterioles, thermal hypoalgesia, tactile allodynia, and intraepidermal nerve fiber loss. All these changes were completely or partially reversed by cariporide treatment. NHE‐1 inhibition was also associated with reduced accumulation of advanced glycation end‐products and nitrated proteins in diabetic peripheral nerve. In conclusion, our findings support an important role for NHE‐1 in PDN, and provide rationale for development of NHE‐1 inhibitors and NHE‐1 inhibitor‐containing combination therapies for treatment of this devastating diabetic complication.
A role for C‐terminal cysteines in oligomerization of the Slc26a6 anion transporter
Authors: Li Qinggang , Babilonia Elisa , Chang Min‐Hwang , Kwon Charles , Mandal Asim , Mercado Adriana , Romero Michael F. , Mount David B.
Published: June 2021

The ten‐member SLC26 gene family encodes anion transporters with a diversity of substrates and transport modes, functioning as electrogenic exchangers, anion sensors, and/or anion channels. Slc26a6 is perhaps the most versatile paralog, mediating electrogenic exchange of multiple divalent and monovalent anions. We have generated a cysteineless Slc26a6 for structure‐function studies, fully substituting 8 cysteines with threonine, serine, and alanine, as dictated by the functional data for single and compound mutants. Although absolute transport rates are reduced in the cysteineless Slc26a6 protein it exhibits equivalent anion specificity and electrophysiology (chloride‐bicarbonate exchange) to that of wild‐type. On Western blots, the oligomerization of wild‐type Slc26a6 is inhibited by dithiothreitol, suggesting a role for inter‐cysteine disulfide bonds; oligomerization is absent in the cysteineless protein. Co‐immunoprecipitation of FLAG‐ and MYC‐tagged Slc26a6 is easily detectable for wild‐type proteins but absent in the epitope‐tagged cysteineless proteins. The equivalent data for single and compound mutants implicate C‐terminal cytoplasmic cysteines in oligomerization of the transporter protein. In summary, the eight cysteines in Slc26a6 are dispensable for transport activity but appear to play a role in oligomerization of the transport protein.
Mapping critical structural elements in divalent metal‐ion transporter‐1 (DMT1)
Authors: Alexander John H , Shawki Ali , Meller Jarek , Mackenzie Bryan
Published: June 2021

DMT1 is essential for nonheme iron absorption and iron utilization by erythroid precursors. Our initial approach towards probing the structure‐function of DMT1 is to (1) predict critical residues based on the results of chemical modification experiments or structural models and (2) test these predictions by site‐directed mutagenesis and functional expression of human DMT1 in RNA‐injected Xenopus oocytes. The results of treatment with a battery of modifying agents point to critical roles for Tyr and Met residues since 55Fe2+ transport (at pH 5.5) was sensitive to Rose Bengal photooxidation (preincubated at pH 9.0), tetranitromethane, and cyanogen bromide. Whereas M240A behaved like wildtype, a M438A mutation inhibited 2 μM 55Fe2+ transport 69 ± 6% (SE), increased 10‐fold the K0.5 for Fe2+ relative to wildtype, and altered substrate recognition such that Mn2+ was excluded (whereas wildtype DMT1 transports Mn2+), suggesting that Met‐438 participates in metal‐ion binding. Further, we postulated that Pro (‘kinked’) residues could be involved in forming a metal‐binding pocket in DMT1. Mutating Pro‐266—selected for its proximity to His‐272, a residue known to comprise part of the H+‐coupling mechanism driving Fe2+ transport [Pflügers Arch Eur J Physiol 451, 544–558 (2006)]—to either Gly (“soft kink”) or Ala (“no kink”) inhibited 55Fe2+ transport 92 ± 3% and 95 ± 3% respectively. We anticipate that this work will begin to reveal structural requirements for metal coordination in DMT1.
ROLE OF KCC3 IN HIGH‐GRADE GLIOMA MIGRATION
Authors: Gagnon Kenneth B. , Delpire Eric
Published: June 2021

Malignant astrocytomas are extremely proliferative and invasive. Tumor migration involves rapid changes in cell volume. Because cation‐chloride cotransporters are involved in cell volume maintenance and regulation, we examined their expression in human brain tumors obtained from the Vanderbilt Molecular Neurosurgical Tissue Bank. We show by quantitative PCR that KCC3 mRNA expression significantly increased between Grade I and Grade IV astrocytomas. In order to investigate the role of KCC3 in cell migration, we used the rat C6 glioma cell model. RT‐PCR demonstrated a similar increase in the mRNA expression level of KCC3 between primary cultured rat astrocytes and C6 glioma cells. Scratch‐wound migration assays demonstrate rapid cell motility leading to wound closure in less time than cell doubling. This migration was prevented by the application of 200 μM furosemide, but not 20 μM bumetanide, indicating participation of K‐Cl cotransport and not Na‐K‐2Cl cotransport. To further assess the role of KCC3 in cell migration, we silenced the expression of the cotransporter with shRNA. Using a clone which demonstrated >80 % reduction in protein expression by western blot analysis and 50% reduction in cotransport activity by tracer flux analysis, we re‐examined the cell motility in a scratch‐wound migration assay. We show shRNA silencing of KCC3 significantly impaired the rate of cell migration.
Facial Muscle Ontogenesis in Human Fetuses With & Without Cleft Lip/palate
Authors: Rogers Carolyn Ruth , Mooney Mark P. , Smith Timothy D. , Marazita Mary L. , Siegel Michael I. , Burrows Anne M.
Published: June 2021

Malformations of the orbicularis oris (OO) muscle in humans with cleft lip and palate have been well characterized. Given their close relation, it stands to reason that other peri‐oral muscles would suffer similar malformations. The purpose of this study was to evaluate the anatomy of these muscles in humans with cleft lip and palate. Stained slides of serially sectioned human fetal heads from individuals with and without cleft lip and palate were examined at 16 and 15.5 weeks of gestational age. The depressor septi (DS), levator labii superioris (LLS), and levator labii superioris alequae nasii (LLSAN) muscles were identified and their cross‐sectional area measured. Average cross‐sectional area was compared on either side in affected and unaffected fetuses. Preliminary results indicate that in the unaffected fetus, average cross‐sectional area (reported as right/left) was: DS (87.7/81.8 um2 ), LLS (153.5/182.3 um2 ), LLSAN (133.8/111.2 um2). For the 15.5 week affected fetus, average cross‐sectional area (reported as affected/unaffected) was: DS (unidentifiable), LLS (33.5/133.0 um2), LLSAN (72.3/96.8 um2). Overall muscle fiber orientation and distribution was disorganized in the area of the cleft, and discrete muscles were difficult to discern. In conclusion, malformations exist in the DS, LLS, and LLSAN muscles in fetuses with cleft lip and palate with overall muscle volume being decreased.
Regulation of podocyte Slo1 BK channels by synaptopodin
Authors: Kim Eun Young , Mundel Peter , Dryer Stuart E
Published: June 2021

Large‐conductance Ca2+‐activated K+ channels encoded by the Slo1 gene are expressed in podocytes, where they can interact with TRPC6 channels at slit diaphragms in close conjunction with cytoskeletal regulatory proteins such as synaptopodin. We observed that synaptopodin and Slo1 proteins can be reciprocally co‐immunoprecipitated when they are heterologously expressed in HEK293T cells. This also occurs with endogenously expressed proteins in an immortalized mouse podocyte cell line, where these proteins are closely co‐localized. GST pull‐down assays show that the both C‐ and N‐terminal domains of synaptopodin can interact with distal portions of cytoplasmic C‐termini of Slo1. Co‐expression of synaptopodin with the VEDEC isoform of Slo1 proteins, which is endogenously expressed in podocytes, increases the expression of Slo1 on the surface of HEK293T cells. Moreover, there is a reduction of Slo1 on the surface of mouse podocytes with stable knockdown of synaptopodin compared to a control cell line. Stable synaptopodin knockdown does not affect total expression of Slo1 proteins but does result in a reduction in macroscopic currents carried by these channels. These data indicate that synaptopodin regulates trafficking of functional BKCa channels in podocytes and thereby modulate responses to mechanical and chemical stimuli. Supported by R01 DK082529 (SED) and RO1 DK057683 (PM)
SGK1 Increases Plasma Membrane CFTR in Human Airway Epithelial Cells
Authors: Bomberger Jennifer , Sato Denry , Coutermarsh Bonita , Barnaby Roxanna , Chapline M Christin , Stanton Bruce
Published: June 2021

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a cyclic AMP‐regulated chloride channel and a regulator of other ion channels and transporters in epithelial tissues. CFTR rapidly cycles between the apical plasma membrane, early endosomes, and recycling endosomes. However, little is known about the regulation of CFTR endocytic trafficking. Thus, understanding the protein‐protein interactions that regulate the intracellular trafficking of CFTR is critical. We recently reported that serum‐ and glucocorticoid‐regulated kinase 1 (SGK1) stimulates CFTR Cl currents in Xenopus oocytes by increasing the number of channels in the plasma membrane. In this study, we examined the effects of SGK1 on CFTR in polarized human airway epithelial cells (WT‐CFTR CFBE41o‐ cells). Dexamethasone (50 nM) rapidly (30–60 minutes) increased SGK1 mRNA and protein abundance in a time‐dependent manner. The increase in SGK1 protein was followed by an increase in CFTR in the apical membrane and total cell lysates. Overexpression of a constitutive‐active SGK, SGK‐S422D, increased apical membrane CFTR, while siRNA knockdown of SGK1 reduced the dexamethasone‐stimulated increase in apical membrane CFTR. Finally, a dominant negative SGK1 (K127N) had no effect on CFTR expression in the apical membrane. These studies demonstrate that SGK1 increases the apical membrane abundance of CFTR in human airway epithelial cells.
Physiological regulation of ENaC in the aldosterone‐sensitive distal nephron by a local endothelin system
Authors: Bugaj Vladislav , Pochynyuk Oleh , Kohan Donald E , Stockand James D
Published: June 2021

We used patch‐clamp electrophysiology to investigate regulation of the epithelial Na+ channel (ENaC) by endothelin‐1 (ET‐1) in isolated, split‐open collecting ducts (CD) from wild type (wt) mice and mice with CD‐specific deletion of ET‐1 (CD ET‐1 −/−) and the ETB receptor (CD ETB −/−). Resting ENaC activity was significantly higher in CD ET‐1 −/− and CD ETB −/− mice compared to wt mice. Moreover, exogenous ET‐1 rapidly and significantly decreases ENaC open probability (Po) in wt and CD ET‐1 −/− but not CD ETB −/− mice. ET‐1 decreases ENaC activity through basolateral membrane ETB receptors. ET‐1 activates src family tyrosine kinases and their downstream MAPK1/2 effector cascade to modulate ENaC. In wt mice, ENaC activity is related inversely to dietary salt intake. This relation is retained but muted in CD ETB −/− mice with these animals requiring higher dietary Na+ intake to achieve the same level of ENaC suppression. We conclude that ENaC activity in mammalian principal cells is decreased in a physiologically relevant manner by activation of a local CD endothelin system where ET‐1 through ETB receptors tempers channel activity, at least in part, through src/MAPK1/2 signaling. These findings provide a mechanism for the natriuresis observed in‐vivo in response to ET‐1, as well as, the salt‐sensitive hypertension caused by disrupting the collecting duct endothelin system.
Developmental changes in the ENaC channel of the chick chorioallantoic membrane
Authors: Gonzalez Laura A. , Silverthorn Dee U
Published: June 2021

Confined to the closed system of the egg, the chick embryo deposits its renal wastes into the chorioallantoic sac, an outgrowth of its hindgut. The allantoic fluid undergoes changes in both volume and ion composition throughout the development of the embryo. These changes are most likely the result of Na+ reabsorption from the allantoic fluid. In previous experiments we demonstrated that short‐circuit current across the epithelia increases five‐fold between embryonic days 9–19. This current is inhibitable by ouabain and amiloride, indicating that Na+‐K+‐ATPase and ENaC channels play a role. Aldosterone increased the amiloride‐inhibitable current from day 9 to day 18, suggesting that the ENaC channels may be up‐regulated by the hormone. The slow (100 min) aldosterone response appears to be the classic gene‐regulated response to a steroid hormone. In this study we are investigating the role of the epithelial sodium channel ENaC in allantoic short‐circuit current. We used RT‐PCR to measure normal developmental changes in the RNA message of the three ENaC subunits in the allantoic epithelium. The RNA message of all subunits increased two‐fold from day 9 to day 11, and then slowly decreased from days 11 to 17. These data were confirmed using Western blots. Subsequent experiments will investigate the role of aldosterone in inducing ENaC protein synthesis.
Functional expression of the epithelial sodium channel delta subunit in human respiratory epithelial cells
Authors: Schwagerus Elena , Buckley Stephen T , Ehrhardt Carsten
Published: June 2021

The expression and function of the epithelial sodium channel δ subunit (δ‐ENaC) as well as its contribution to ion and fluid transport in human respiratory epithelial cells.

Expression of δ‐ENaC was investigated in human alveolar (A549) and bronchial (Calu‐3, 16HBE14o‐) epithelial cell lines on genomic and protein levels. Reverse transcriptase and real‐time PCR were carried out as well as analysis by Western blot and immuno‐confocal laser scanning microscopy (CLSM). Ussing chamber studies were performed with Calu‐3 monolayers using Evans blue at 10 – 50 μM.

Delta‐ENaC expression was confirmed in all cell lines on mRNA level with higher abundance in A549 and 16HBE14o‐ than in Calu‐3 cell monolayers. Generally, mRNA levels of δ‐ENaC were weaker than that of α, β and γ ENaC subunits. By Western blot and CLSM δ‐ENaC protein was detected in all cell lines, with the highest expression found in Calu‐3 and 16HBE14o‐ cells. CLSM also revealed the δ‐ENaC subunit located in the apical aspect of cellular membranes. Evans blue exhibited a concentration‐dependant increase of ISC up to 650% at 50 μM.

These results indicate that ä‐ENaC is functionally expressed in human respiratory epithelial cell monolayers. Further experiments will investigate its role in ion and fluid transport processes across the respiratory epithelial barrier.

ES is funded by an IRSCET postgraduate scholarship.
Phosphopeptide Screen Uncovers JNK1 as a Potentiator of Nedd4‐2‐Mediated Epithelial Na+ Channel Inhibition
Authors: Bhalla Vivek , Oyster Nicholas M , Wijngaarden Marjolein , Lee Jeffrey , Li Hui , Xia Xiaoyu , Huang Zhirong , Chalkley Robert , Burlingame Alma , Pearce David , Hallows Ken
Published: June 2021

The ubiquitin ligase Nedd4‐2 inhibits several ion transport proteins including the epithelial Na+ channel (ENaC). To define new regulatory inputs to Nedd4‐2 function, we screened for novel sites of Nedd4‐2 phosphorylation using tandem mass spectrometry. Three of seven identified Nedd4‐2 Ser/Thr phosphorylation sites corresponded to previously identified target sites for serum‐and‐glucocorticoid kinase 1 (SGK1), while four were novel, including Ser‐293, which matched the consensus for a mitogen‐activated protein kinase (MAPK) target sequence. Nedd4‐2 was found to be a target of c‐Jun N‐terminal kinase 1 (JNK1), but not of p38‐MAPK or extracellular‐regulated kinase (ERK1/2). Further rounds of tandem mass spectrometry identified two additional JNK1‐phosphorylated residues within Nedd4‐2: Thr‐408 and Thr‐899. Mutations at all three JNK1 sites markedly inhibited JNK1‐dependent phosphorylation, ENaC‐inhibitory activity by Nedd4‐2, and Nedd4‐2 ubiquitin ligase activity. Together, these data establish JNK1 as a novel potentiator of Nedd4‐2, which may work in conjunction with inhibitory kinases (e.g. SGK1 and PKA) to govern Nedd4‐2 regulation of epithelial ion transport. Supported by the NIH NIDDK.
Regulation of ENaC by proprotein convertase subtilisin/kexin type 9(PCSK9)
Authors: Sharotri Vikas , Snyder Peter M
Published: June 2021

The epithelial Na+channel ENaC forms a pathway for Na+ absorption across epithelia. Various proteases regulate ENaC by altering its activity. Here we tested the effect of the serine protease proprotein convertase subtilisin/kexin type 9 (PCSK9) on ENaC. We found that PCSK9 and ENaC interact using a coimmunoprecipitation assay. PCSK9 decreased amiloride sensitive Na+ current in Xenopus oocytes and in FRT epithelia expressing αβγENaC. This resulted from decreased ENaC cell surface expression. Using a cycloheximide chase assay we found that PCSK9 accelerated degradation of ENaC. The proteasomal inhibitor ALLN reversed this decrease in ENaC protein. In contrast, PCSK9 had no effect on endocytosis or degradation of the cell surface pool of ENaC. Together, the data indicate that PCSK9 regulates ENaC by increasing its degradation in the biosynthetic pathway.

Supported by funding from American Heart Association 0920147G (VS) and NIH HL058812 (PMS)
An SP/TP motif in the N‐terminal cytoplasmic domain of UT‐A3 as potential activation site of UT‐A3‐mediated urea transport
Authors: Mistry Abinash , Mallick Rickta , Klein Janet D. , Sands Jeff M. , Fröhlich Otto
Published: June 2021

The UT‐A protein variant UT‐A3 possesses in its N‐terminal cytoplasmic domain two SP sites, which are potential phosphorylation sites by proline‐directed kinases. In rat UT‐A3, they are located at positions 15/16 and 35/36. The purpose of the experiments was to test if these sites are involved in the cAMP‐dependent activation of urea influx in UT‐A3‐expressing Xenopus oocytes. By mutating two consensus phosphorylation sites for protein kinase A (S85 and S92 of mouse UT‐A3), Smith and coworkers (Smith CP et al, AJP Cell 287:C1087, 2004) had shown that these PKA sites were not responsible for the observed cAMP dependence of UT‐A3 in oocytes. We prepared N‐terminal deletion constructs of UT‐A3 in which the deleted sequence was replaced by a start codon. Thus these constructs started with a methionine either at positions 18 (del18), 35 (del35) or 53 (del53). When expressed in Xenopus oocytes, UT‐A3‐del18 had the same urea transport activity and cAMP stimulation as wild‐type UT‐A3. However, UT‐A3‐del35 and UT‐A3‐del53, while exhibiting the same unstimulated urea transport activity, were not stimulated by cAMP treatment. To test the hypothesis that the SP35 site is involved in the activation of UT‐A3, we mutated the S15 and S35 either alone or together. As expected, UT‐A3‐S15A behaved like wild‐type UT‐A3 whereas S35A and S15A/S35A were no longer stimulated by cAMP treatment. These data suggest that SP35 is responsible for the activation of UT‐A3 in oocytes. They are also consistent with the findings of the proteomic study of Hoffert et al. (PNAS 103:7159, 2006) who found that S35 was phosphorylated in response to vasopressin in the renal inner medulla. This research was supported by NIH grant DK41707 and the Emory University Research Council
Effects of cytochrome P450 metabolites of arachidonic acid on the epithelial sodium channel (ENaC)
Authors: Pavlov Tengis S , Ilatovskaya Daria , Roman Richard J , Staruschenko Alexander
Published: June 2021

Sodium reabsorption via ENaC in mammalian distal nephron plays a central role in body fluid volume regulation. It was previously shown that arachidonic acid (AA) significantly decreased ENaC activity in cortical collecting duct (CCD) cells and that effect of AA is mediated by 11,12‐EET. CYP450 metabolites of AA have been implicated in the control of sodium and water excretion. LC/MS/MS analysis revealed the presence of EET‐8,9, EET‐11,12, EET‐14,15 and 5‐HETE, 12/8‐HETE, 15‐HETE, but not 20‐HETE in cultured renal collecting duct principal mpkCCDc14 cells. Coexpression of Cyp2c8 but not Cyp4a10 with all three ENaC subunits in CHO cells significantly decreased amiloride‐sensitive current density in whole‐cell experiments. Single channel analysis revealed that EET‐8,9, EET‐11,12 and EET‐14,15 acutely decrease ENaC activity in the mpkCCDc14 cells. In contrast, neither 5‐HETE nor 12/8‐HETE or 15‐HETE have effect on ENaC activity. We conclude that EET‐8,9, EET‐11,12 and EET‐14,15 are important for maintaining transepithelial Na+ transport and ENaC activity.

Supported by ASN, AHA and MCW Research Affairs Committee.
Interaction Of COMMD Family Proteins With The Epithelial Sodium Channel.
Authors: Liu Yongfeng , Swart Marianne , McDonald Fiona
Published: June 2021

The COMMD (Copper Metabolism gene Murr1 Domain) family of proteins consists of 10 members, COMMD1‐10. The prototype of the COMMD family, COMMD1, was found to be involved in copper metabolism, NF‐κB signaling and sodium uptake. The classic epithelial sodium channel (ENaC) in the distal kidney is made up of three similar subunits, α, β and γENaC subunits. Another ENaC family member, δENaC, is expressed throughout the brain. Previously our laboratory identified COMMD1 was an ENaC‐interacting protein and inhibited amiloride‐sensitive Na+ transport of the δβγENaC subunits and αβγENaC channels [1].

Therefore, we hypothesized other COMMD proteins might (1) bind to ENaC, (2) regulate ENaC and (3) if other COMMD proteins do regulate ENaC activities, COMMD might be involved in the Nedd4‐2 ubiquitination pathway in the regulation of ENaC. Our GST pulldown and co‐immunoprecipitation results have shown that most COMMD proteins bound to α, β, γ and δENaC subunits. Using immunohistochemistry experiments we show that COMMD3 and COMMD9 are both expressed in kidney and overlay with αENaC expression. Functional data from Ussing chamber experiments has shown that COMMD3 and COMMD9 down‐regulate ENaC currents in FRT cells. Therefore COMMD3 and COMMD9 may be novel regulators of ENaC.
The Molecular Chaperones, ERdj3 and ERdj4, Two Hsp40s, Select ENaC for ER Associated Degradation (ERAD)
Authors: Boyd Cary Ragan , Buck Teresa M , Kolb Alexander R , Kleyman Thomas R , Brodsky Jeffrey L
Published: June 2021

The epithelial Na+ channel (ENaC) is a vital channel and mutations in ENaC may contribute to hyper‐ and hypotension and cystic fibrosis. ENaC consists of 3 homologous subunits (α, β, γ) that assemble in the ER and traffic to the plasma membrane. ENaC assembly is inefficient with <20% reaching the plasma membrane, and a large fraction of ENaC subunits are targeted for ER‐associated degradation (ERAD). Commonly, selection of ERAD substrates occurs via the chaperones Hsp70s and Hsp40s; however, little is known about the chaperones involved in the ERAD of ENaC. We therefore employed a yeast expression system and identified two Hsp40s, Jem1 and Scj1, that function as ENaC chaperones. The mammalian homologs of these Hsp40s are ERdj3 and ERdj4, and our hypothesis was that ERdj3 and ERdj4 over‐expression would increase ENaC degradation and cause a decrease in ENaC function‐expression. Using a Xenopus oocyte expression system and two‐electrode voltage clamp (TEV), we discovered that ENaC current was reduced >30% by co‐expression of ERdj3 or ERdj4. This reduction in current was proteosome‐mediated and correlated with a decrease in ENaC surface expression. However, these Hsp40s neither affected the rate of ENaC endocytosis nor exocytosis. These data support the hypothesis that Hsp40s are important in the ERAD of ENaC, and that data obtained in our yeast system can be recapitulated in a vertebrate‐expression system. Funded by NIH: GM75061, DK65161, and DK79307
ATF3 is activated in ventilator induced lung injury
Authors: Dos Santos Claudia , Akram Ali , Masoom Hussain , Han Bing , Haitsma Jack J. , Peng Claudia , Shan Yuexin , Zhang Haibo , Liu Mingyao , Hai Tsonwin , Slutsky Arthur S.
Published: June 2021

Introduction

Mechanical ventilation (MV) has been shown to cause ventilator‐induced lung injury (VILI). We wanted to identify novel molecular targets involved in reducing the harmful effects of MV (VILI).

Methods

To identify potential novel genes and proteins involved in VILI we i) performed microarray analysis on previously published VILI animal model data, ii) exposed human bronchial epithelial cells (Beas‐2b) in vitro to mechanical cyclic stretch (30 cycles/min for 4 hours), iii) performed western blot analysis on these stretched and non‐stretched cells, and iv) validated the regulation of the newly discovered gene(s) in vivo using wild type (WT) and ATF3 (Activator transcription factor 3) knock‐out mice (ATF3 KO). These two groups of mice were exposed to either injurious or non‐injurious ventilation strategies in combination with LPS or saline.

Results

ATF3 was identified by microarray analysis as one of the main genes involved in VILI. The presence of this factor was validated in vitro in the mechanically cyclic‐stretched Beas‐2b cells. Western blot assays confirmed expression of ATF3 in the stretched cells and their absence in the static cells. We further confirmed the regulation of ATF3 using WT and ATF3 KO mice in a model of VILI in vivo. ATF3 KO mice exposed to LPS in combination with injurious MV had more inflammation than WT mice of similar treatment or mice treated with saline.

Conclusion

Expression of ATF3 is stretch dependent and this gene seems to confer protection in combination with MV. Specific targeting of this factor in clinical settings could reduce VILI in patients receiving MV.
Mechanisms of Zinc Oxide Nanoparticle (ZONP)‐Induced Alveolar Epithelial Cell (AEC) Injury
Authors: KIM YONG HO , FAZLOLLAHI FARNOOSH , YACOBI NAZANIN R , BOROK ZEA , KIM KWANG‐JIN , CRANDALL EDWARD D
Published: June 2021

We investigated ZONP (20 nm diameter) induction of AEC injury utilizing primary rat alveolar epithelial cell monolayers (RAECM). We assessed injury by measuring transmonolayer resistance (RT) and equivalent short‐circuit current (IEQ), lactate dehydrogenase (LDH) release, intracellular reactive oxygen species (ROS) and mitochondrial activity. We also investigated effects of free Zn2+ released from ZONP on RT and IEQ of RAECM. Results showed that apical exposure to 176 μg/mL ZONP decreased RT and IEQ of RAECM by 100% over 24h, whereas exposure to 11 μg/mL ZONP had little effect. Apical exposure of RAECM for 24h to 176 μg/mL ZONP caused irreversible injury. ZONP effects on RT yielded a half‐maximal concentration of 17 μg/mL. About 8 μg/mL of Zn2+ was found to be released from 176 μg/mL ZONP suspension over 24h. When an equivalent concentration of Zn2+ (as ZnCl2) or supernatant from 24h ZONP suspensions was used to apically expose RAECM, RT was decreased by ~45% after 24h. Apical exposure for 24h to 176 μg/mL ZONP increased LDH release, disrupted cell plasma membrane integrity, decreased mitochondrial activity and increased intracellular ROS production. These data indicate that apical ZONP exposure of RAECM leads to severe cellular injury involving increased oxidative stress and disruption of cell membrane integrity, effects due in part to free Zn2+ released from ZONP. (Funded by: NIH; Hastings & Whittier Foundations.)
Protein kinase activation is required for secretagogue‐stimulated membrane‐association of fusion promoting protein annexin A7 in alveolar type II cells
Authors: Chander Avinash , Gerelsaikhan Tudevdagva , Holbrook Kelly , Hayrapetyan Yedema
Published: June 2021

The secretion of lung surfactant in alveolar type II (T2) cells requires fusion of lamellar bodies (LB) with the plasma membrane. Our in vitro studies have shown a role for Annexin A7 (A7) in such membrane fusion, since purified A7 binds to LB and plasma membranes and promotes their fusion. Using antibodies against recombinant A7, we have shown that secretagogues of lung surfactant promote membrane‐association of endogenous A7 in isolated T2 cells. We now show that activation of protein kinase is required for such membrane‐association. Isolated rat T2 cells were treated for 30min with phorbol myristate acetate (PMA), A23187, ATP or isoproterenol, and the A7 levels in the cytosol and membrane fractions were analyzed by immuno‐blot analysis. All agents increased relative proportion of A7 in the membranes when compared with the control cells. Immuno‐fluorescence staining of T2 cells showed that secretagogues promoted relocation of A7 to membranes including LB, as verified by co‐staining for ABCA3. Because of the significance of protein kinase activation in regulation of surfactant secretion, we evaluated the effect of protein kinase inhibitors on A7 relocation in treated cells. The PMA‐induced relocation was inhibited by pretreatment with bis‐indolylmaleimide I, a specific inhibitor of PKC, and the isoproterenol‐induced relocation was blocked with H‐89, a specific inhibitor of PKA. Thus, our studies provide further correlative evidence for the role of A7 in lung surfactant secretion.
Mitochondrial donation by mesenchymal stromal cells rescues alveolar surfactant secretion in sepsis
Authors: Islam Mohammad Naimul , Otsu Keishi , Houser Sandra D. , Lindert Jens , Bhattacharya Jahar
Published: June 2021

In sepsis, bone marrow derived mesenchymal stromal cells (MSCs) protect against acute lung injury (ALI). However, underlying mechanisms are unclear. We reported, MSCs transfer mitochondria to cultured capillaries (Otsu et al, 2008). To determine whether mitochondrial transfer occurs in situ, in anesthetized rats we intratracheally instilled lipopolysaccharide (LPS) or saline. After 4 hours, we instilled MSCs (2×106 cells) that expressed the mitochondria‐targeted fluorescent protein, DsRed. After 24 hours, we isolated and blood‐perfused the lungs. In alveoli viewed by confocal microscopy, we determined DsRed fluorescence and alveolar surfactant secretion in single type 2 (AT2) cells (Ashino et al, 2000). In saline‐treated lungs, no alveolar MSCs were evident. In LPS‐treated lungs, in some but not all alveoli MSCs adhered to alveolar epithelium (AE) and DsRed localized intracellularly in AT2 cells. Surfactant secretion stimulated by a 15‐second hyperinflation, was evident in alveoli containing MSCs, but not in MSC‐free alveoli. Our findings indicate that in LPS‐treated lungs (i) MSCs transfer mitochondria to AT2 cells, and that (ii) surfactant secretion is abrogated in MSC‐free alveoli, but protected in alveoli containing MSCs. MSC‐AEC mitochondrial transfer might increase ATP in AT2 cells, thereby rescuing surfactant secretion and protecting lung function in ALI (support HL64896 & HL57556).
Mechanisms of Apical Fluid Volume Control in Airway Epithelia
Authors: Falkenberg Cibele Vieira , Jakobsson Eric
Published: June 2021

Our objective is to use computer modeling to understand mechanisms of apical fluid volume control in an airway epithelium. We choose human bronchial epithelium (HBE) because of clinical importance. To build the model, we utilize published short and open circuit electrical and pharmacological data. We use a stochastic parametric search tool to establish parameters for transporters and channels in the tissue, namely permeabilities for Na, K, Cl and HCO3 and the HK pump in the apical membrane, and permeabilities for K and Cl, plus the NaK pump, NKCC, NBC, AE and the NHE in basolateral membrane. To complete the model, we postulate that the NKCC is sensitive to cell volume and the apical Pna permeability (ENaC) is sensitive to the apical volume. Important results include: a) with active regulation of cell and apical volumes by basolateral NKCC and apical ENaC the system assumes an oscillating steady state. Absent active regulation, the system drifts to “fluid catastrophe” either flooding or drying out, and b) if the apical Pcl is changed, the apical Pna necessary to maintain the apical fluid thickness varies such that the ratio of Pna/Pcl is maintained roughly constant. Supported by NIH 5PN2EY016570
Functional expression of the hypo‐osmosensitive channels TRPV4 and TRPM3 in mouse airway epithelium
Authors: Lembrechts Robrecht , Brouns Inge , Schnorbusch Kathy , Pintelon Isabel , Timmermans Jean‐Pierre , Adriaensen Dirk
Published: June 2021

TRPV4 and TRPM3, members of the transient receptor potential channel family, are membrane proteins that mediate Ca2+ entry in cells upon application of hypo‐osmotic solutions. This study aimed at investigating the expression and functionality of these osmosensitive channels in the epithelium of mouse intrapulmonary airways by combining confocal Ca2+ imaging and multilabel immunohistochemistry.

Immunostaining revealed that TRPV4 and TRPM3 are colocalized on both ciliated cells and Clara cells, the most common cell types in the mouse airway epithelium, but also that the neuroendocrine cells, grouped as neuroepithelial bodies, are negative. Confocal live cell imaging of precision cut vibratome slices of mouse lungs loaded with Fluo‐4 showed that short term (30s) perfusion with a hypo‐osmotic solution (200 mOsm) results in a clear and reversible rise in intracellular Ca2+ in all Clara and ciliated cells.

Both TRPV4 and TRPM3 channels may allow the cells to react to changes in extracellular osmolarity and therefore potentially play a central role in airway epithelial homeostasis by modulating epithelial water transport. The presented approach opens interesting new perspectives for unraveling functional aspects of diverse cell and tissue types in control lungs and disease models.

Support: IWT fellowship SB/81162 (R.L); FWO grant G.0081.08 (D.A.); UA grants GOA BOF 2007 (D.A.) and KP BOF 2006 (I.B.)
Age and sex differences in the hypercapnic ventilatory response in awake neonatal rats
Authors: Wenninger Julie M , Holley Heidi S , Behan Mary
Published: June 2021

Sex hormones exert powerful effects in the central nervous system during development. Little is known about sex differences in the development of chemoreceptors and their contribution to sexual dimorphism in respiratory diseases such as Sudden Infant Death Syndrome (SIDS) and Obstructive Sleep Apnea (OSA). Several chemo sensitive brainstem nuclei, including the caudal raphe, undergo structural and functional changes in early postnatal life. The purpose of this study was to determine whether there are sex differences in the ventilatory response to hypercapnia (FICO2 = 7%) in rats from P10‐P15, and whether there are sex‐related changes in 5‐HT1A receptor expression in the caudal raphe in these male and female rat pups. Awake neonatal rats were studied each day in a plethysmograph in normoxia and CO2. Arterial O2 saturation was measured with a pulse oximeter. After the study, a blood sample was drawn for measurement of sex hormone levels. The animals were perfused and brainstems harvested for immunohistochemical evaluation. During CO2 exposure we found sex differences in tidal volume (VT) and minute ventilation (VE) at postnatal days 11 and 15. There were also significant changes in breathing frequency, VT, and VE with age in each sex. These data suggest that there are sex differences in the hypercapnic ventilatory response in neonatal rats during a critical period of ventilatory development. Supported by NIH AG18760 and Parker B. Francis Foundation.
Chorioamnionitis decreases neonatal respiratory pattern variability in transverse rat brainstem slices
Authors: Mayer Catherine Ann , Balan Kannan , Kc Prabha , Martin Richard , Wilson Christopher G
Published: June 2021

Chorioamnionitis is a major cause of perinatal inflammation and infection, and has been implicated in brain morbidity associated with preterm birth. Apnea of prematurity is a common clinical manifestation of neonatal inflammation suggesting that chorioamnionitis has a direct effect on the development of central respiratory output which is detectable using non‐linear analyses. We modeled chorioamnionitis by injecting 150 μg/kg of lipopolysaccharide (LPS) into the chorion space in alternate sacs (30 μl/sac) of pregnant Sprague Dawley dams (E16). Following normal birth of the pups, on the second postnatal day, we cut 500 μm transverse brainstem slices from LPS exposed and sham pups and recorded fictive inspiratory output from the hypoglossal nerve (XII) rootlet at both 5 and 8mM external [K+]. We analyzed the XII signal to quantify burst duration, interburst interval, and area of XII burst. We found no effect of prenatal LPS exposure on Te, Ti, frequency or coefficient of variation at either K+ concentration. However, the histogram entropy of the interburst intervals (a measure of variability), was significantly lower at 5mM K+ in LPS exposed pups (3.5±1.3) than shams (5.08±0.7). This suggests that endotoxin exposure in prenatal life impairs inspiratory pattern generation and such abnormalities could lead to ventilatory abnormalities in postnatal life.