Functional interaction of the K-Cl cotransporter  (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant ( K i) ∼190 μM] and pretreatment with NEM ( K i ∼60 μM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]iin activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.

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Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00462.2006 ◽

2007 ◽

Vol 293 (5) ◽

pp. F1699-F1713 ◽

Cited By ~ 112

Author(s):

Ling Wu ◽

Xiaochong Gao ◽

Rachel C. Brown ◽

Stefan Heller ◽

Roger G. O'Neil

Keyword(s):

Shear Stress ◽

Calcium Influx ◽

Collecting Duct ◽

Loss Of Function ◽

Western Blots ◽

Hek 293 ◽

Hek 293 Cells ◽

Hek Cells ◽

293 Cells

Gain/loss of function studies were utilized to assess the potential role of the endogenous vanilloid receptor TRPV4 as a sensor of flow and osmolality in M-1 collecting duct cells (CCD). TRPV4 mRNA and protein were detectable in M-1 cells and stably transfected HEK-293 cells, where the protein occurred as a glycosylated doublet on Western blots. Immunofluorescence imaging demonstrated expression of TRPV4 at the cell membranes of TRPV4-transfected HEK and M-1 cells and at the luminal membrane of mouse kidney CCD. By using intracellular calcium imaging techniques, calcium influx was monitored in cells grown on coverslips. Application of known activators of TRPV4, including 4α-PDD and hypotonic medium, induced strong calcium influx in M-1 cells and TRPV4-transfected HEK-293 cells but not in nontransfected cells. Applying increased flow/shear stress in a parallel plate chamber induced calcium influx in both M-1 and TRPV4-transfected HEK cells but not in nontransfected HEK cells. Furthermore, in loss-of-function studies employing small interference (si)RNA knockdown techniques, transfection of both M-1 and TRPV4-transfected HEK cells with siRNA specific for TRPV4, but not an inappropriate siRNA, led to a time-dependent decrease in TRPV4 expression that was accompanied by a loss of stimuli-induced calcium influx to flow and hypotonicity. It is concluded that TRPV4 displays a mechanosensitive nature with activation properties consistent with a molecular sensor of both fluid flow (or shear stress) and osmolality, or a component of a sensor complex, in flow-sensitive renal CCD.

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Cadmium affects osmotic phase and regulatory volume decrease in cultured human embryonic kidney cells

Journal of Biological Research - Bollettino della Società Italiana di Biologia Sperimentale ◽

10.4081/jbr.2016.5650 ◽

2016 ◽

Vol 89 (1) ◽

Author(s):

Rossana Morabito ◽

Alessia Remigante ◽

Roberta Costa ◽

Silvia Dossena ◽

Giuseppa La Spada ◽

...

Keyword(s):

Cell Volume ◽

Regulatory Volume Decrease ◽

Cell Swelling ◽

Suitable Model ◽

Human Embryonic Kidney ◽

Cell Homeostasis ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Volume Decrease

The present investigation aims to verify whether cadmium (Cd2+), a metal possibly accumulated in body tissues from air and food, affects cell volume regulation capability in cultured human embryonic kidney (HEK 293 Phoenix) cells. The osmotic phase (OP), which is the expected cell swelling due to aquaporins involvement after hyposmotic challenge, and regulatory volume decrease (RVD), bringing cell volume back to control values through Ca2+-dependent ion efflux (K+ and Cl–), have been monitored in HEK 293 cells treated with Cd2+ (1-10-100 μM) for different time intervals (30 min, 3 h, overnight) and then submitted to 15 % hyposmotic shock. The results show that both 1 and 10 μM Cd2+ significantly reduced OP, whereas 100 μM impaired Cd2+ RVD mechanisms. The use of glutathione (GSH, 200 μM) confirmed that Cd2+ elicited its effect via oxidative damage, being RVD inhibition after Cd2+ treatment prevented by this antioxidant compound. Our findings show that: i) HEK 293 cells are a suitable model to assay the effect of xenobiotics on cell homeostasis; ii) Cd2+, depending on its concentration, affects cell homeostasis at different levels, i.e. water and ion permeability, responsible for, respectively, OP and RVD mechanism, adding thus more information to the knowledge of Cd2+ toxicology.

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Rare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function

AJP Renal Physiology ◽

10.1152/ajprenal.00552.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. F840-F847 ◽

Cited By ~ 36

Author(s):

Michelle Y. Monette ◽

Jesse Rinehart ◽

Richard P. Lifton ◽

Biff Forbush

Keyword(s):

Blood Pressure ◽

Thick Ascending Limb ◽

Transport Function ◽

Lower Blood Pressure ◽

Wild Type ◽

Hek 293 ◽

Hek 293 Cells ◽

Hek Cells ◽

293 Cells ◽

Lower Blood

The Na-K-Cl cotransporter (NKCC2) is the major salt transport pathway in the thick ascending limb of Henle's loop and is part of the molecular mechanism for blood pressure regulation. Recent screening of ∼3,000 members of the Framingham Heart Study identified nine rare independent mutations in the gene encoding NKCC2 (SLC12A1) associated with clinically reduced blood pressure and protection from hypertension (Ji WZ, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State M, Levy D, Lifton RP. Nat Genet 40: 592–599, 2008). To investigate their functional consequences, we introduced the nine mutations in human NKCC2A and examined protein function, expression, localization, regulation, and ion transport kinetics using heterologous expression in Xenopus laevis oocytes and HEK-293 cells. When expressed in oocytes, four of the mutants (T235M, R302W, L505V, and P569H) exhibited reduced transport function compared with wild-type. In HEK-293 cells, the same four mutants exhibited reduced function, and in addition N399S and P1083A had significantly lower activity than wild-type. The two most functionally impaired mutants (R302W and L505V) exhibited dramatically diminished production of complex-glycosylated protein and a decrease in or absence of plasma membrane localization, indicative of a processing defect. All of the functional human (h) NKCC2A variants were regulated by changes in oocyte volume and intracellular chloride in HEK cells, but P254A and N399S exhibited a lower constitutive activity in HEK cells. The P569H mutant exhibited a 50% reduction in sodium affinity compared with wild-type, predicting lower transport activity at lower intratubular salt concentrations, while the P254A mutant exhibited a 35% increase in rubidium affinity. We conclude that defects in NKCC2 processing, transport turnover rate, regulation, and ion affinity contribute to impaired transport function in six of the nine identified mutants, providing support for the predictive approach of Ji et al. to identify functionally important residues by sequence conservation. Such mutations in hNKCC2A are likely to reduce renal salt reabsorption, providing a mechanism for lower blood pressure.

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Functional Interaction Between Caveolin 1 and LRRC8-Mediated Volume-Regulated Anion Channel

Frontiers in Physiology ◽

10.3389/fphys.2021.691045 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mikel Rezola ◽

Aida Castellanos ◽

Xavier Gasull ◽

Núria Comes

Keyword(s):

Glutamate Release ◽

Anion Channel ◽

Cell Swelling ◽

Physical Interaction ◽

Biophysical Properties ◽

Hek 293 ◽

Caveolin 1 ◽

Hek 293 Cells ◽

293 Cells ◽

Essential Subunit

Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5′-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl– currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.

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Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽

10.4161/auto.25455 ◽

2013 ◽

Vol 9 (9) ◽

pp. 1407-1417 ◽

Cited By ~ 41

Author(s):

Patience Musiwaro ◽

Matthew Smith ◽

Maria Manifava ◽

Simon A. Walker ◽

Nicholas T. Ktistakis

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Anesthesiology ◽

10.1097/00000542-200512000-00009 ◽

2005 ◽

Vol 103 (6) ◽

pp. 1156-1166 ◽

Cited By ~ 7

Author(s):

Kevin J. Gingrich ◽

Son Tran ◽

Igor M. Nikonorov ◽

Thomas J. Blanck

Keyword(s):

Charge Transfer ◽

Calcium Channels ◽

Dependent Manner ◽

Current Time ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Dependent Inactivation ◽

Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

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