scholarly journals The expression of wild-type pendrin (SLC26A4) in human embryonic kidney (HEK 293 Phoenix) cells leads to the activation of cationic currents

2005 ◽  
Vol 153 (5) ◽  
pp. 693-699 ◽  
Author(s):  
Silvia Dossena ◽  
Antonella Maccagni ◽  
Valeria Vezzoli ◽  
Claudia Bazzini ◽  
Maria Lisa Garavaglia ◽  
...  
Keyword(s):  
Chloride Transport ◽  
Cellular Systems ◽  
Wild Type ◽  
Human Embryonic Kidney ◽  
Pendred Syndrome ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Hek 293 ◽  
Prelingual Deafness ◽  
Open Question

Objective: The SLC26A4 protein (pendrin) seems to be involved in the exchange of chloride with other anions, therefore being responsible for iodide organification in the thyroid gland and the conditioning of the endolymphatic fluid in the inner ear. Malfunction of SLC26A4 leads to Pendred syndrome, characterized by mild thyroid dysfunction often associated with goiter and/or prelingual deafness. The precise function of the SLC26A4 protein, however, is still elusive. An open question is still whether the SLC26A4-induced ion exchange mechanism is electrogenic or electroneutral. Recently, it has been shown that human pendrin expressed in monkey cells leads to chloride currents. Methods: We overexpressed the human SLC26A4 isoform in HEK293 Phoenix cells and measured cationic and anionic currents by the patch-clamp technique in whole cell configuration. Results: Here we show that human pendrin expressed in human cells does not lead to the activation of chloride currents, but, in contrast, leads to an increase of cationic currents. Conclusion: Our experiments suggest that the SLC26A4-induced chloride transport is electroneutral when expressed in human cellular systems.

Basal chloride currents in murine airway epithelial cells: modulation by CFTR

1998 ◽  
Vol 274 (4) ◽  
pp. C904-C913 ◽  
Author(s):  
R. Tarran ◽  
M. A. Gray ◽  
M. J. Evans ◽  
W. H. Colledge ◽  
R. Ratcliff ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Wild Type ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Cation Permeability ◽  
Current Voltage ◽  
Respiratory Cells

We have isolated ciliated respiratory cells from the nasal epithelium of wild-type and cystic fibrosis (CF) null mice and used the patch-clamp technique to investigate their basal conductances. Current-clamp experiments on unstimulated cells indicated the presence of K+ and Cl− conductances and, under certain conditions, a small Na+conductance. Voltage-clamp experiments revealed three distinct Cl− conductances. I tv-indep was time and voltage independent with a linear current-voltage ( I- V) plot; I v-actexhibited activation at potentials greater than ±50 mV, giving an S-shaped I- Vplot; and I hyp-act was activated by hyperpolarizing potentials and had an inwardly rectified I- Vplot. The current density sequence was I hyp-act = I v-act ≫ I tv-indep. These conductances had Cl−-to- N-methyl-d-glucamine cation permeability ratios of between 2.8 and 10.3 and were unaffected by tamoxifen, flufenamate, glibenclamide, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited by Zn2+ and Gd3+. I tv-indep and I v-act were present in wild-type and CF cells at equal density and frequency. However, I hyp-actwas detected in only 3% of CF cells compared with 26% of wild-type cells, suggesting that this conductance may be modulated by cystic fibrosis transmembrane conductance regulator (CFTR).

scholarly journals Bupivacaine inhibits a small conductance calcium‐activated potassium type 2 channel in human embryonic kidney 293 cells

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Hongfei Chen ◽  
Zhousheng Jin ◽  
Fangfang Xia ◽  
Zhijian Fu
Keyword(s):  
Free Calcium ◽  
Heart Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background Bupivacaine blocks many ion channels in the heart muscle, causing severe cardiotoxicity. Small-conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unclear. This study investigated the effect of bupivacaine on SK2 channels. Methods The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch-clamp technique was used to examine the effect of bupivacaine on SK2 channels. The concentration–response relationship of bupivacaine for inhibiting SK2 currents (0 mV) was fitted to a Hill equation, and the half-maximal inhibitory concentration (IC50) value was determined. Results Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine, and lidocaine on SK2 currents was 16.5, 46.5, and 77.8µM, respectively. The degree of SK2 current inhibition by bupivacaine depended on the intracellular concentration of free calcium. Conclusions The results of this study suggested the inhibitory effect of bupivacaine on SK2 channels. Future studies should explore the effects of SK2 on bupivacaine cardiotoxicity.

scholarly journals Kinetic, Mechanistic, and Structural Aspects of Unliganded Gating of Acetylcholine Receptor Channels

2000 ◽  
Vol 115 (5) ◽  
pp. 621-635 ◽  
Author(s):  
Claudio Grosman ◽  
Anthony Auerbach
Keyword(s):  
Equilibrium Constant ◽  
Spontaneous Activity ◽  
Acetylcholine Receptor ◽  
Reaction Pathway ◽  
Good Description ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Patch Clamp Technique ◽  
Mouse Muscle ◽  
Hek 293

The spontaneous activity of adult mouse muscle acetylcholine receptor channels, transiently expressed in HEK-293 cells, was studied with the patch-clamp technique. To increase the frequency of unliganded openings, mutations at the 12′ position of the second transmembrane segment were engineered. Our results indicate that: (a) in both wild type and mutants, a C ↔ O kinetic scheme provides a good description of spontaneous gating. In the case of some mutant constructs, however, additional states were needed to improve the fit to the data. Similar additional states were also needed in one of six patches containing wild-type acetylcholine receptor channels; (b) the δ12′ residue makes a more pronounced contribution to unliganded gating than the homologous residues of the α, β, and ε subunits; (c) combinations of second transmembrane segment 12′ mutations in the four different subunits appear to have cumulative effects; (d) the volume of the side chain at δ12′ is relevant because residues larger than the wild-type Ser increase spontaneous gating; (e) the voltage dependence of the unliganded gating equilibrium constant is the same as that of diliganded gating, but the voltage dependences of the opening and closing rate constants are opposite (this indicates that the reaction pathway connecting the closed and open states of the receptor changes upon ligation); (f) engineering binding-site mutations that decrease diliganded gating (αY93F, αY190W, and αD200N) reduces spontaneous activity as well (this suggests that even in the absence of ligand the opening of the channel is accompanied by a conformational change at the binding sites); and (g) the diliganded gating equilibrium constant is also increased by the 12′ mutations. Such increase is independent of the particular ligand used as the agonist, which suggests that these mutations affect mostly the isomerization step, having little, if any, effect on the ligand-affinity ratio.

scholarly journals Residues 248–252 and 300–304 of the cardiac Na+/Ca2+ exchanger are involved in its regulation by phospholemman

2011 ◽  
Vol 301 (4) ◽  
pp. C833-C840 ◽  
Author(s):  
Xue-Qian Zhang ◽  
JuFang Wang ◽  
Jianliang Song ◽  
Angi M. Ji ◽  
Tung O. Chan ◽  
...  
Keyword(s):  
Deletion Mutants ◽  
Partial Reduction ◽  
Wild Type ◽  
Human Embryonic Kidney ◽  
Intracellular Loop ◽  
Hek 293 ◽  
Interaction Sites ◽  
Empty Vector ◽  
293 Cells ◽  
Linker Domain

Using split cardiac Na+/Ca2+ exchangers (NCX1), we previously demonstrated that phospholemman (PLM) regulates NCX1 by interacting with the proximal linker domain (residues 218–358) of the intracellular loop of NCX1. With the use of overlapping loop deletion mutants, interaction sites are localized to two regions spanning residues 238–270 and residues 300–328 of NCX1. In this study, we used alanine (Ala) linker scanning to pinpoint the residues in the proximal linker domain involved in regulation of NCX1 by PLM. Transfection of human embryonic kidney (HEK)293 cells with wild-type (WT) NCX1 or its Ala mutants but not empty vector resulted in NCX1 current ( INaCa). Coexpression of PLM with WT NCX1 inhibited INaCa. Mutating residues 248–252 (PASKT) or 300–304 (QKHPD) in WT NCX1 to Ala resulted in loss of inhibition of INaCa by PLM. By contrast, inhibition of INaCa by PLM was preserved when residues 238–242, 243–247, 253–257, 258–262, 263–267, 305–309, 310–314, 315–319, 320–324, or 325–329 were mutated to Ala. While mutating residue 301 to alanine completely abolished PLM inhibition, mutation of any single residue 250–252, 300, or 302–304 resulted in partial reduction in inhibition. Mutating residues 248–252 to Ala resulted in significantly weaker association with PLM. The NCX1-G503P mutant that lacks Ca2+-dependent activation retained its sensitivity to PLM. We conclude that residues 248–252 and 300–304 in the proximal linker domain of NCX1 were involved in its inhibition by PLM.

scholarly journals Evaluation of cytotoxic profile of hydroalcoholic extract of fruit rinds of Garcinia pedunculata on human embryonic kidney and breast carcinoma cells

2020 ◽  
Vol 9 (2) ◽  
pp. 259
Author(s):  
Sukrant Sharma ◽  
Ravi Mundugaru ◽  
Pradeepa H. Dakappa ◽  
Pundalik R. Naik
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cytotoxic Effect ◽  
Metastatic Breast ◽  
Cancer Cell Line ◽  
Cancer Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Alcoholic Extract ◽  
Human Embryonic Kidney ◽  
Hek 293

Background: The fruit rinds of Garcinia pedunculata has potential medicinal properties and used in many chronic ailments. It has been demonstrated that cytoprotective effects in various experimental research works. But its cytotoxic effect has not been evaluated. The present study was aimed to screen its relative cytotoxic effect on normal and cancer cell lines.Methods: In the present study, the cytotoxic effect of hydro alcoholic extract of Garcinia pedunculata was evaluated on normal human embryonic kidney (HEK-293) and M.D. Anderson metastatic breast cancer cell lines (MDA-MB 231) using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.Results: Higher dose level of hydro alcoholic extract of Garcinia pedunculata (HAGP) (500 μg/ml) has shown considerable increase (112.503) in the percentage viability of HEK-29 whereas; there is a remarkable decrease in the viable cell population (77.490) in MDA-MB 231.Conclusions: Based on the observed results we could conclude that HAGP has potential cytotoxic effect on the cancer cell line without altering the normal cell growth and proliferation. Thus it has potential to develop as a safer chemotherapeutic agent. Further detailed exploration is required to confirm its therapeutic efficacy in different cancer cell lines.

scholarly journals SLC26A9 is a constitutively active, CFTR-regulated anion conductance in human bronchial epithelia

2009 ◽  
Vol 133 (4) ◽  
pp. 421-438 ◽  
Author(s):  
Carol A. Bertrand ◽  
Ruilin Zhang ◽  
Joseph M. Pilewski ◽  
Raymond A. Frizzell
Keyword(s):  
Current Increase ◽  
Chloride Currents ◽  
Hek 293 ◽  
Anion Secretion ◽  
Anion Conductance ◽  
Hek Cells ◽  
Δf508 Cftr ◽  
Cf Transmembrane Conductance Regulator ◽  
Apical Membranes ◽  
In Cells

Human bronchial epithelial (HBE) cells exhibit constitutive anion secretion that is absent in cells from cystic fibrosis (CF) patients. The identity of this conductance is unknown, but SLC26A9, a member of the SLC26 family of CF transmembrane conductance regulator (CFTR)-interacting transporters, is found in the human airway and exhibits chloride channel behavior. We sought differences in the properties of SLC26A9 and CFTR expressed in HEK 293 (HEK) cells as a fingerprint to identify HBE apical anion conductances. HEK cells expressing SLC26A9 displayed a constitutive chloride current that was inhibited by the CFTR blocker GlyH-101 (71 ± 4%, 50 µM) and exhibited a near-linear current–voltage (I-V) relation during block, while GlyH-101–inhibited wild-type (wt)CFTR exhibited a strong inward-rectified (IR) I-V relation. We tested polarized HBE cells endogenously expressing either wt or ΔF508-CFTR for similar activity. After electrical isolation of the apical membrane using basolateral α-toxin permeabilization, wtCFTR monolayers displayed constitutive chloride currents that were inhibited by GlyH-101 (68 ± 6%) while maintaining a near-linear I-V relation. In the absence of blocker, the addition of forskolin stimulated a current increase having a linear I-V; GlyH-101 blocked 69 ± 7% of the current and shifted the I-V relation IR, consistent with CFTR activation. HEK cells coexpressing SLC26A9 and wtCFTR displayed similar properties, as well as forskolin-stimulated currents that exceeded the sum of those in cells separately expressing SLC26A9 or wtCFTR, and an I-V relation during GlyH-101 inhibition that was moderately IR, indicating that SLC26A9 contributed to the stimulated current. HBE cells from CF patients expressed SLC26A9 mRNA, but no constitutive chloride currents. HEK cells coexpressing SLC26A9 with ΔF508-CFTR also failed to exhibit SLC26A9 current. We conclude that SLC26A9 functions as an anion conductance in the apical membranes of HBE cells, it contributes to transepithelial chloride currents under basal and cAMP/protein kinase A–stimulated conditions, and its activity in HBE cells requires functional CFTR.

In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP

1997 ◽  
Vol 273 (5) ◽  
pp. L1065-L1072 ◽  
Author(s):  
Thomas J. Kelley ◽  
Calvin U. Cotton ◽  
Mitchell L. Drumm
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Nasal Epithelium ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cyclic Monophosphate ◽  
Δf508 Cftr ◽  
Membrane Bound

Inhibitors of guanosine 3′,5′-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl− transport across the nasal epithelia of cystic fibrosis mice carrying the ΔF508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (ΔF/ΔF)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and ΔF508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(−/−)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 ± 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3′,5′-cyclic monophosphate (6.2 ± 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl− secretion across the nasal epithelium of CFTR(ΔF/ΔF) mice. No effect on TEPD was seen with this combination when used on CFTR(−/−) mice, implying that the CNP-induced change in TEPD in CFTR(ΔF/ΔF) mice is CFTR dependent.

scholarly journals Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains

2001 ◽  
Vol 281 (1) ◽  
pp. C248-C256 ◽  
Author(s):  
Dominique Trouet ◽  
Iris Carton ◽  
Diane Hermans ◽  
Guy Droogmans ◽  
Bernd Nilius ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Inhibitory Effect ◽  
Kinase Domain ◽  
Wild Type ◽  
Patch Clamp Technique ◽  
Src Tyrosine Kinase ◽  
Src Homology ◽  
Cpae Cells ◽  
Src Homology Domains ◽  
Homology Domains

We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosine kinase on I Cl,swell, the swelling-induced Cl−current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect I Cl,swell. However, transfection of c-Src with a Ser3Cys mutation that introduces a dual acylation signal and targets c-Src to lipid rafts and caveolae strongly repressed hypotonicity-induced I Cl,swell in CPAE cells. Kinase activity was dispensable for the inhibition of I Cl,swell, since kinase-deficient c-Src Ser3Cys either with an inactivating point mutation in the kinase domain or with the entire kinase domain deleted still suppressed VRAC activity. Again, the Ser3Cys mutation was required to obtain maximal inhibition by the kinase-deleted c-Src. In contrast, the inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude that c-Src-mediated inhibition of VRAC requires compartmentalization of c-Src to caveolae and that the Src homology domains 2 and/or 3 are necessary and sufficient for inhibition.

scholarly journals Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

2020 ◽  
pp. 175342592092747
Author(s):  
Yajie Yang ◽  
Yan Hu ◽  
Yile Zhou ◽  
Tao Liang ◽  
Haihong Tang ◽  
...  
Keyword(s):  
Inflammatory Factors ◽  
Myeloid Differentiation ◽  
Study Group ◽  
Tlr4 Expression ◽  
Wild Type ◽  
Human Embryonic Kidney ◽  
Gram Negative ◽  
Tnf Α ◽  
Polymorphic Variants ◽  
Myeloid Differentiation Factor

TLR4 polymorphisms such as Asp299Gly and Thr399Ile related to Gram-negative sepsis have been reported to result in significantly blunted responsiveness to LPS. Our study group previously screened other TLR4 polymorphic variants by checking the NF-κB activation in comparison to wild type (WT) TLR4 in human embryonic kidney 293T cells. In this study, we found that the Lys694Arg (K694R) polymorphism reduced the activation of NF-κB, and the production of downstream inflammatory factors IL-1, TNF-α and IL-6, representing the K694R polymorphism, led to blunted responsiveness to LPS. Then, we examined the influence of the K694R polymorphism on total and cell-surface TLR4 expression by Western blotting and flow cytometry, respectively, but observed no differences between the K694R polymorphism and WT TLR4. We also used co-immunoprecipitation to determine the interaction of the K694R polymorphism and WT TLR4 with their co-receptor myeloid differentiation factor 2 (MD2) and their downstream signal adaptor MyD88. We found that K694R reduced the recruitment of MyD88 in TLR4 signalling but had no impact on the interaction with MD2.

Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

2007 ◽  
Vol 292 (3) ◽  
pp. F1028-F1034 ◽  
Author(s):  
W. Bruce Sneddon ◽  
Yanmei Yang ◽  
Jianming Ba ◽  
Lisa M. Harinstein ◽  
Peter A. Friedman
Keyword(s):  
Conditioned Media ◽  
Kidney Cells ◽  
Wild Type ◽  
Egfr Transactivation ◽  
Hek 293 ◽  
Erk Activation ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Extracellular Signal

The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of Gi, which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.

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