bluuebunny/crossref_metadata_2025_split huggingface dataset:
Discrepancy between TRPA1 activation by reversible and irreversible electrophiles suggests involvement of cytosolic cofactors other than polyphosphates.
Authors: Parks Thomas A , Bahia Parmvir K , Taylor‐Clark Tom 
Published: November 2023

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel that is expressed in a subset of nociceptive afferent sensory nerves. When activated, TRPA1 can evoke nociceptive responses (e.g. pain and defensive reflexes), as well as peripheral neurogenic inflammation. TRPA1 is activated by a host of endogenous (inflammatory mediators) and exogenous (pollutants, food) agonists. Many TRPA1 agonists are electrophilic compounds that covalently modify reactive cysteine residues on TRPA1, leading to activation of the channel. Electrophiles can bind through reversible or irreversible reactions based on the structure of the compound. Here, patch clamp techniques and calcium imaging have been used to characterize human TRPA1 (hTRPA1) channel activity (expressed in HEK293 cells) in the presence of reversible and irreversible electrophiles. Calcium imaging shows that both reversible electrophiles (e.g. allylisothiocyanate [AITC]) and irreversible electrophiles (e.g. iodoacetamide [IA] and N‐ethylmaleimide [NEM]) rapidly induce TRPA1 activation. In contrast, our patch clamp studies show a discrepancy between irreversible and reversible electrophilic TRPA1 activation. Whole cell patch clamp studies exhibit a reduction in the kinetics and magnitude of TRPA1 activation by IA and NEM compared to AITC. Furthermore, single channel patch clamp experiments display reduced open probability with IA and NEM compared to AITC. Both whole cell patch and excised patch clamp recordings were performed in the presence of 5mM polyphosphates. In preliminary gramicidin perforated patch recordings there was no difference in NEM and AITC‐evoked TRPA1 activation. Thus in intact cellular assays (Ca2+ imaging and gramicidin‐perforated patch) there is little difference in TRPA1 activation by reversible and irreversible electrophiles, whereas in dialyzed cellular assays (whole cell patch and excised patch) irreversible electrophiles evoked reduced TRPA1 activation compared to reversible electrophiles. The data suggests that cytosolic factors other than polyphosphates are important for TRPA1 activation by irreversible electrophilic ligands.Support or Funding InformationNHLBI R01 (HL119802) and Supplement (HL119802‐S1)
The Role of Pore‐Lining Residues of MEC‐4 and MEC‐10 in the Channel's Response to Shear Stress
Authors: Shi Shujie , Kleyman Thomas R. 
Published: November 2023

In the nematode Caenorhabditis elegans, gentle body touch is sensed via a multi‐protein mechanotransduction apparatus expressed in the worm's touch receptor neurons. At the core of this mechanotransduction apparatus is a mechanosensitive ion channel formed by two homologous protein, MEC‐4 and MEC‐10. Like other members of the epithelial Na+ channel (ENaC)/degenerin family, the channel's pore is primarily lined by the second transmembrane helixes (TM2) of MEC‐4 and MEC‐10. Strikingly, there are nine and four touch‐insensitive mutations within the MEC‐4 TM2 and MEC‐10 TM2, respectively, highlighting the importance of the pore‐lining residues in transmitting gating signals.When expressed in Xenopus oocytes, the MEC‐4/MEC‐10 channel is activated by flow‐mediated laminar shear stress (LSS), and both MEC‐4 and MEC‐10 are required for a robust LSS response. To explore the role of the pore‐lining residues of MEC‐4 and MEC‐10 in the channel's regulation by mechanical forces, we introduced point mutations at the specific sites within the MEC‐4 TM2 or MEC‐10 TM2 and examined its response to LSS in Xenopus oocytes. We observed that the LSS response was largely diminished by touch‐insensitive mec‐10 alleles that introduce charged residues into the channel's pore. Both the magnitude and kinetics of channel action by LSS were altered when mutations were introduced at specific sites within MEC‐4 TM2. In addition, we found that channels with high intrinsic Po (MEC‐4 A713D, or MEC‐4 A713C modified with MTSES) showed reduced response to LSS. Together, our results support an essential role of the pore‐forming regions of MEC‐4 and MEC‐10 in channel gating in response to mechanical stimuli.Support or Funding InformationThis work was financially supported, in whole or in part by National Institutes of Health Grants: DK103834 (to S.S.), DK051391 (to T.R.K.), and DK079307 (Pittsburgh Center for Kidney Research)
SCN3A‐encoded Voltage‐gated Sodium Channel NaV1.3 is Specifically Expressed in Human and Mouse Gastrointestinal Enterochromaffin Cells and is Important for Enterochromaffin Cell Excitability
Authors: Strege Peter R. , Knutson Kaitlyn R. , Eggers Samuel J , Wang Fan , Li Joyce , Leiter Andrew , Farrugia Gianrico , Beyder Arthur 
Published: November 2023

BACKGROUNDIn the gastrointestinal (GI) epithelium, enterochromaffin (EC) cells are enteroendocrine cells that use tryptophan hydroxylase 1 (TPH‐1) to produce serotonin (5‐hydroxytryptamine, 5‐HT). EC cells release 5‐HT in response chemical and mechanical stimuli. EC cell 5‐HT affects GI secretion, motility and sensation. Other neuroendocrine cells are electrically excitable, and several types use SCN3A‐encoded voltage‐gated sodium channel NaV1.3 for electrical excitability. It is unknown whether EC cells are electrically excitable.AIMTo determine if the EC cells are electrically excitable.METHODSMurine TPH1‐CFP small bowel was dissociated, FAC sorted for TPH1‐CFP cells, and ion channel expression was analyzed by RNA‐seq. Murine TPH1‐CFP and human small bowel and colon were analyzed by immunohistochemistry (IHC). After dissociation, primary cultures from TPH1‐CFP mouse jejunum and colon were maintained for 72 hours. Scn3a expression in CFP+, CFP− cultured cells and media was analyzed by single cell polymerase chain reaction (PCR). Voltage‐ and current‐clamp experiments were performed on TPH1‐CFP cells at 24–48 hours.RESULTSRNA‐seq of FACS TPH1‐CFP EC cells from murine small bowel showed that Scn3a was the 3rd most densely expressed ion channel gene, and no other ScnXa genes showed significant expression. Scn3a encodes a voltage‐gated sodium channel NaV1.3. By IHC, NaV1.3 is expressed in ~89–93% of the human and mouse small bowel and colon EC cells, which were defined as TPH1‐CFP+ for mouse and 5‐HT+ for human. Single cell PCR of CFP+ cells from primary cultures confirmed Scn3a expression. In voltage‐clamp, we found voltage‐gated fast activating and inactivating voltage‐gated currents in TPH1‐CFP EC cells from murine small bowel (78% of cells, −86±16 pA/pF, n=28), and colon (65% of cells, −66±12 pA/pF, n=14). These currents were abolished by Na+ substitution by NMDG+ and blocked by the NaV1.3 inhibitor ICA‐121431 (IC50 = 131±53 nM). Current‐clamped EC cells showed both elicited and spontaneous action potentials, also abolished by Na+ substitution and NaV1.3 block by ICA‐121431. Spontaneous current‐clamp activity showed action potential bursts of variable amplitude and frequency.CONCLUSIONSSCN3A‐encoded voltage‐gated sodium channel NaV1.3 is densely and specifically expressed by the EC cells of the human and mouse small and large bowel. In mouse EC cells, NaV1.3 generates voltage‐gated sodium current which is critical for EC cell electrical excitability.Support or Funding InformationNIH DK106456, DK84567, DK052766
Involvement of Intracellular Transport in Regulation of TREK‐1 Channel in 293T Cells
Authors: Andharia Naaz , Joseph Ancy , Hayashi Mikio , Okada Masayoshi , Matsuda Hiroko 
Published: November 2023

IntroductionPotassium (K+) channels play a crucial roles in the regulation of the resting membrane potential and excitability of various cells. The mammalian two‐pore domain K+ channels are responsible for background or leak K+ currents, one of which is TREK‐1 (TWIK‐related K+ channel). The current of TREK‐1 channel appears to be regulated by intracellular transport, in which the cytosolic C‐terminus has been shown to play a important role in cell surface expression of the channel. An interesting feature of the TREK‐1 channel is the run‐up: i.e. the current through TREK‐1 channels spontaneously increases within several minutes in the whole‐cell patch‐clamp mode. Since C‐terminal domain is required for the surface localization of the TREK‐1 channel, the run‐up might be regulated by transport to the plasma membrane.ObjectivesThe present study aimed to investigate the involvement of intracellular transport in the run‐up.MethodsWe stably transduced TREK‐1c or its fusion protein with mCherry to the 293T cells and measured whole‐cell currents with patch‐clamp techniques. Cell surface localization was analyzed with confocal microscopic analysis and protein biotinylation.ResultsInhibitors of membrane protein transport, N‐methylmaleimide (NEM) and brefeldin‐A, inhibited the run‐up, while an endocytosis inhibitor, pitstop2, facilitated the run‐up. Correspondingly, in cells stably expressing the mCherry‐TREK‐1c fusion protein, NEM decreased the surface localization of mCherry‐TREK‐1c, whereas pitstop2 increased it. The run‐up was inhibited by the intracellular application of a peptide of the C‐terminal fragment, TREK335‐360, corresponding to the interaction site with microtubule associated protein 2 (Mtap2). The extracellular application of an Ezrin inhibitor (NSC668394) also suppressed the surface localization and the run‐up. The co‐application of these inhibitors abolished the TREK‐1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK‐1c channels and run‐up.ConclusionThese results indicate that intracellular protein transport is involved in the run‐up of TREK‐1c currents.COI: NOKey words: K+ channel, TREK‐1, intracellular transport, microtubule‐associated protein 2, ezrinSupport or Funding InformationThis study was supported by KAKENHI from the JSPS, the SICP from the JST, the Heiwa Nakajima Foundation, the Ryobi Memorial Foundation (to MO), the Mishima Memorial Foundation, the Rotary Yoneyama Memorial Foundation (to NA), and the Fujii Memorial Foundation (to AJ).
Regulation of claudins in renal TJ by Na+,K+‐ATPase alpha 1 subunit
Authors: Larre Maria Isabel , Zhang Jue , Xie Zijian 
Published: November 2023

Tight junctions (TJs) are located at the uppermost portion of the lateral membrane of epithelial cells that seal the intracellular interspace between adjacent cells, transforming the epithelial cells into an effective permeable barrier. Tight junctions regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. In the kidneys, the molecular composition of TJ determines the permeability and ion selectivity of different nephron segments along renal tubules. For example, while proximal tubules express claudin‐2, distal nephrons express the claudin‐4 isoform. Little is known about what controls the composition of TJ along the nephron. Previous reports have shown that ouabain, a cardiac glycoside that acts by inhibiting the Na+,K+‐ATPase pump activity, increases TJ sealing degree (10–100 nM), suggesting the participation of Na+,K+‐ATPase in TJ regulation. In this study, with newly developed rescue α1 mutant protocol, we were able to generate unique stable epithelial cell lines that express Na+,K+‐ATPase alpha‐1 with specific mutations in the N domain. The expression of Na+,K+‐ATPase alpha‐1 mutants (A425P and A420P) does not affect the activity of sodium pump but increases the level expression of Src phosphorylation at Y418 and also decrees the expression of caveolin‐1. This study is the first approach to investigated the role of Na+,K+‐ATPase alpha 1 on TJ protein expression and function on kidney epithelial cells.ResultsUsing TER measurement, western blot and confocal microscopy we found that A420P and A425P mutation decreases the ion permeability and changes the molecular composition of TJ proteins when compared to wild type Na+,K+‐ATPase alpha‐1. The expression levels and cellular localization of Claudin‐2 and claudin‐4 were altered. Src inhibitor PP2 and pNaKtide were able to decrease the TER values in A425P cell line comparable to wild type. Thus, this work shows that the N domain of Na+/K+‐ATPase alpha‐1 has an important role in tight junction function and this regulation implicates Na+,K+‐ATPase‐Src‐Caveolin‐Claudin pathway. Taken together, these observations have a significant relevance to the altered function of kidneys in physiological and pathological conditions, in which the transport of ion and nutrients are significantly altered.
The membrane trafficking and functionality of the K+‐Cl− co‐transporter KCC2 is regulated by TGF‐β2
Authors: Khakipoor Shokoufeh , Roussa Eleni 
Published: November 2023

GABAA response shifts from depolarizing to hyperpolarizing during neuronal maturation and it is well established that K+‐Cl−‐cotransporter KCC2 up‐regulation is responsible for this process. Failure in KCC2 up‐regulation is seen in epilepsy and stroke. Surprisingly, there is not much known about regulatory pathways involved in the KCC2 expression, trafficking, and functional expression. Here, we investigate the effect of transforming growth factor beta 2, TGF‐β2, in the regulation of KCC2 trafficking and functional expression in mouse mature hippocampal neurons and analyze the underlying molecular pathways. Therefore embryonic day 18 primary hippocampal neurons and hindbrain slices were used. KCC2 protein and surface expression is investigated by immunoblotting, surface biotinylation and immunofluorescence. Signaling pathways are studied using siRNA knockdown, co‐immunoprecipitation, and 3D STED microscopy. KCC2 functional expression is measured by somatodendritic Cl− gradient recording via whole cell patch clamp. The results show that KCC2 surface and functional expression is significantly increased in hippocampal neurons treated with TGF‐β2, compared to non‐treated cells. TGF‐β2 treatment leads to cAMP response element‐binding protein (CREB) phosphorylation and Ras‐associated binding protein 11b (Rab11b) gene expression and also increases Rab11b/KCC2 interaction and co‐localization. Knocking down either CREB or Rab11b suppresses KCC2 membrane and functional expression. Pre‐Bötzinger complex neurons in TGF‐β2 deficient mice have a significant decrease in KCC2 membrane expression, compared to wildtype and exogenous TGFβ2 application can rescue this phenotype. We introduce TGF‐β2/CREB/Rab11b as a novel regulatory pathway involved in KCC2 trafficking and functional activity in mouse mature hippocampal neurons.Support or Funding InformationThis work was supported by grants from the Deutsche Forschungsgemeinschaft [grant numbers KR1477/15‐1, SFB 780 TP B02 to K.K. and E.R.].
Functional analysis of a novel pathogenic mutation affecting the gating glutamate of the 2Cl−/H+ exchanger ClC‐5 in patients with Dent's disease type 1
Authors: Bignon Yohan , Lahuna Olivier , Frachon Nadia , Teulon Jacques , Vargas‐Poussou Rosa , Lourdel Stéphane 
Published: November 2023

Dent's disease is an X‐linked recessive renal tubular disorder characterized by low‐molecular‐weight proteinuria, hypercalciuria, nephrolithiasis, nephrocalcinosis, and progressive renal failure, inconstantly associated with other proximal tubular dysfunctions. Inactivating mutations of CLCN5, the gene encoding the 2Cl−/H+ exchanger ClC‐5, are associated with Dent's disease type 1. In the kidney, ClC‐5 is predominantly expressed at the apical membrane of proximal tubule cells and on early endosomes. Impaired endosomal acidification consecutive to ClC‐5 loss‐of‐function might explain the defective endocytosis in proximal tubule cells observed in patients with Dent's disease.Here, we have investigated a novel ClC‐5 pathogenic missense mutation (E211G) affecting the gating glutamate that is responsible for coupling the Cl‐flux to the H+ counter‐transport in ClC 2Cl−/H+ exchangers. The courants were characterized using two‐electrode voltage‐clamp recordings in X. laevis oocytes. ClC‐5 subcellular localization was investigated in X. laevis oocytes using luminescence assays and in HEK293T cells using immunofluorescence. Protein expression was characterized in HEK293T cells using western‐blot.The E211G mutant displayed a nearly linear current/voltage relationship. In contrast to those of wild‐type ClC‐5, currents of the mutant were not sensitive to extracellular acidification. We found no difference between this mutant and wild‐type ClC‐5 in terms of protein expression and subcellular targeting. The mutant trafficked normally to the cell surface and to the early endosomes and displayed complex glycosylation.The present study demonstrates that the E211G mutation considerably affects electrical activity of ClC‐5 in proximal tubule cells.Support or Funding InformationThis work was supported in part by grant “Prix Jeune Chercheur” from “La Fondation du Rein”.
Phenotypic consequences of genetic deletion of the glial circadian clock Bmal1 in mice
Authors: Kofuji Paulo , Massman Logan , Engeland William 
Published: November 2023

Glial cells have emerged as key players in brain information processing given the extensive neuronal‐glial and glial‐neuronal communication that takes place at synapses in the central nervous system. Glial cells also express an intrinsic molecular clock and release the neuromodulator ATP in a circadian fashion. Also disruption of glial signaling in fruit flies severely disrupts behavioral circadian rhythms. Collectively, these data suggest that glial cells and their intrinsic circadian rhythms have an important role in modulating circadian rhythmicity. In an effort to link glial cell‐specific circadian clocks to organismal circadian rhythmicity, we studied mice in which the canonical clock gene Aryl hydrocarbon receptor nuclear translocator‐like protein 1 (Bmal1) is selectively disrupted in astrocytes (GFAP‐Bmal1‐cKO). Staining of brain sections for BMAL1 reveals loss of expression in astrocytes, but not in neurons. By crossing GFAP‐Bmal1‐cKO mice with the Per2Luc knock‐in mice in which the clock gene Per2 is fused with the reporter gene Luciferase, we performed real time luminescence analysis of Per2 gene expression from cultured astrocytes and explant tissues. Cultured astrocytes from GFAP‐Bmal1‐cKO/ Per2Luc mice show reduced PER2Luc rhythmicity in comparison with control cultured astrocytes. Ex vivo explants from GFAP‐Bmal1‐cKO/Per2Luc mice of the suprachiasmatic nucleus, adrenal gland, cornea, lung, liver, and pituitary exhibited robust circadian rhythms of PER2Luc rhythmicity. Running wheel activity from GFAP‐Bmal1‐cKO mice subjected to a 12:12h light‐dark cycle reveals locomotor activity that was largely indistinguishable from wild‐type controls. In constant darkness, some mutant mice exhibited faster free running periods. Finally, some mutant mice also exhibited abnormal entrainment to a skeleton photoperiod. These experiments suggest an important role of the intrinsic circadian clock in glial cells for behavioral rhythmicity.Support or Funding InformationRO3 NS3NS094419
Auto‐Immunity Targeting the Enteric Nervous System May Cause Constipation in Multiple Sclerosis
Authors: Spear Estelle T , Haag Melody M , Lavoie Brigitte , Applebee Angela , Teuscher Cory , Mawe Gary M 
Published: November 2023

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that is primarily associated with somatic motor, sensory, and visual deficits. This disease can also cause gastrointestinal (GI) dysmotility, including constipation, but the etiology of this symptom in MS is poorly understood. Additionally, patients with MS typically have serum antibodies against CNS myelin and neuronal proteins that are also found in the enteric nervous system (ENS). Here, we test the hypothesis that constipation in MS is caused by auto‐antibodies that target the ENS. First, we investigated whether serum from MS patients contains antibodies that are immunoreactive against ENS targets of guinea pig myenteric plexus. We found that MS serum (n=20) targets ENS cells more intensely than age and gender matched healthy serum (n=20) (p<0.05), with stained structures including enteric neurons and glia. Next, we tested whether mice with experimental autoimmune encephalomyelitis (EAE) exhibit signs of constipation by assessing whole GI transit, colonic propulsive motility, and fecal consistency. EAE was induced in C57BL/6 or SJL/J mice by immunization against mouse spinal cord homogenate. Whole GI transit was slower in EAE SJL mice than controls (p<0.05) but was not different in EAE C57 mice. Both SJL and C57 mice with EAE had significantly slower colonic transit than control mice of the same background (C57 & SJL p<0.05). The percentage of fecal water content was significantly lower in SJL and C57 EAE mice compared to controls (C57 p<0.01, SJL p<0.0001). To test whether antibodies play a role in GI dysmotility in EAE, we assessed motility in B cell deficient EAE mice (muMt− mice). Colonic transit time was slower in EAE muMt− mice compared to muMt− control mice (p<0.05), but there was no difference in fecal water content. In summary, these data demonstrate that serum antibodies in MS patients target the ENS and could cause constipation. Further, the EAE mouse model of MS has disrupted GI motility, and this effect is partially ameliorated in B cell deficient EAE mice. Together, these data suggest that GI dysmotility in MS involves an autoimmune mediated disruption of enteric neuro‐glial function.Support or Funding InformationSupported by a pilot grant from the NMSS, and a predoctoral fellowship to ETS from NIDDK/NIH.
Nω‐Nitro‐L‐Arginine Methyl Ester (L‐NAME) induced hypertension and cardiorenal oxidative stress: Modulatory effect of the methanolic extract of Azadirachta indica
Authors: Omobowale Temidayo Olutayo , Oyagbemi Ademola , Ugbor Fisayo , Adejumobi Olumuyiwa , Adedapo Adeolu , Yakubu Momoh 
Published: November 2023

Hypertension, a sustained elevation of blood pressure is known to cause both structural and functional abnormalities in both the cardiovascular and renal systems. In this study, we investigated the modulatory effects of the methanol extract of the leaves of Azadirachta indica (AI) on L‐NAME‐induced hypertension and cardiorenal dysfunction via oxidative stress in rats.Fifty rats divided into five (5) groups each containing 10 animals were used in this study. Group A (Control) received only clean tap water for twenty‐one (21) days. Group B was treated orally with L‐NAME alone at the dosage of 40 mg/kg for 21 days. Groups C and D were treated orally with the methanol extract of AI at 100 mg/kg and 200 mg/kg, respectively together with L‐Name for 21 days, while Group E received Enalapril at the dosage of 25 mg/kg alongside L‐NAME for 21 days. Systolic, diastolic and mean arterial pressures were recorded in conscious animals before the termination of the experiment by tail cuff plethysmography. The animals were thereafter fasted overnight, sacrificed and the hearts and kidneys were removed from the animals in each group. Biochemical assays to assess the antioxidant defence system and oxidative stress markers were carried out on cardiac and renal tissues.Treatment with L‐NAME led to a significant (p <0.05) increase in blood pressure and markers of oxidative stress in cardiac and renal tissues. AI caused an improvement in antioxidant defence systems, reduction in markers of oxidative stress and the restoration of blood pressure comparable with that of Enalapril.This study suggests that the methanol extract of AI possesses modulatory functions in L‐NAME induced hypertension and cardiorenal oxidative stress.Support or Funding InformationNone