scholarly journals Rescue of Volume-regulated Anion Current by Bestrophin Mutants with Altered Charge Selectivity

2008 ◽  
Vol 132 (5) ◽  
pp. 537-546 ◽  
Author(s):  
Li-Ting Chien ◽  
H. Criss Hartzell
Keyword(s):  
Mammalian Cells ◽  
Anion Channel ◽  
Peritoneal Macrophages ◽  
Hek293 Cells ◽  
S2 Cells ◽  
Wild Type ◽  
Biophysical Properties ◽  
Ionic Selectivity ◽  
Charge Selectivity ◽  
Cl Channels

Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca2+-activated Cl− channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca2+-activated Cl− currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES−) (PCs/PCl = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs+ than Cl−. The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES− treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1−/−/best2−/−). VRACs were identical in wild-type and best1−/−/best2−/− mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

Both the wild type and a functional isoform of CFTR are expressed in kidney

1996 ◽  
Vol 270 (6) ◽  
pp. F1038-F1048 ◽  
Author(s):  
M. M. Morales ◽  
T. P. Carroll ◽  
T. Morita ◽  
E. M. Schwiebert ◽  
O. Devuyst ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Transmembrane Domain ◽  
Renal Medulla ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Binding Domains ◽  
Cl Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

scholarly journals Volume-regulated Cl− current: contributions of distinct Cl− channels and localized Ca2+ signals

2019 ◽  
Vol 317 (3) ◽  
pp. C466-C480 ◽  
Author(s):  
Yani Liu ◽  
Huiran Zhang ◽  
Hongchao Men ◽  
Yuwei Du ◽  
Ziqian Xiao ◽  
...  
Keyword(s):  
Anion Channel ◽  
Fluorescent Imaging ◽  
Hek293 Cells ◽  
Cell Swelling ◽  
Anoctamin 1 ◽  
Component Cell ◽  
Intracellular Ca ◽  
Cl Channels ◽  
A Cell ◽  
Necessary And Sufficient

The swelling-activated chloride current ( ICl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of ICl,swell is the volume-regulated anion channel (VRAC). Leucine-rich repeat containing 8A (LRRC8A; SWELL1) was recently identified as an essential component of VRAC, but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl− channels, such as anoctamin 1 (ANO1), were also suggested to contribute to ICl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of ICl,swell; we also explored the role of intracellular Ca2+ in ICl,swell activation. We used a CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology, and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to ICl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca2+ transients, and these Ca2+ signals were required to activate both the LRRC8A- and ANO1-dependent components of ICl,swell. Both ICl,swell components required localized rather than global Ca2+ for activation. Interestingly, while intracellular Ca2+ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca2+ transients linked to the ICl,swell activation were mediated by the G protein-coupled receptor-independent PLC isoforms.

scholarly journals Drosophila Bestrophin-1 Chloride Current Is Dually Regulated by Calcium and Cell Volume

2007 ◽  
Vol 130 (5) ◽  
pp. 513-524 ◽  
Author(s):  
Li-Ting Chien ◽  
H. Criss Hartzell
Keyword(s):  
Cell Volume ◽  
Anion Channel ◽  
Cell Swelling ◽  
Macular Dystrophy ◽  
Dependent Manner ◽  
S2 Cells ◽  
New Family ◽  
Drosophila S2 Cells ◽  
Cl Channels ◽  
Dose Dependent

Mutations in the human bestrophin-1 (hBest1) gene are responsible for Best vitelliform macular dystrophy, however the mechanisms leading to retinal degeneration have not yet been determined because the function of the bestrophin protein is not fully understood. Bestrophins have been proposed to comprise a new family of Cl− channels that are activated by Ca2+. While the regulation of bestrophin currents has focused on intracellular Ca2+, little is known about other pathways/mechanisms that may also regulate bestrophin currents. Here we show that Cl− currents in Drosophila S2 cells, that we have previously shown are mediated by bestrophins, are dually regulated by Ca2+ and cell volume. The bestrophin Cl− currents were activated in a dose-dependent manner by osmotic pressure differences between the internal and external solutions. The increase in the current was accompanied by cell swelling. The volume-regulated Cl− current was abolished by treating cells with each of four different RNAi constructs that reduced dBest1 expression. The volume-regulated current was rescued by transfecting with dBest1. Furthermore, cells not expressing dBest1 were severely depressed in their ability to regulate their cell volume. Volume regulation and Ca2+ regulation can occur independently of one another: the volume-regulated current was activated in the complete absence of Ca2+ and the Ca2+-activated current was activated independently of alterations in cell volume. These two pathways of bestrophin channel activation can interact; intracellular Ca2+ potentiates the magnitude of the current activated by changes in cell volume. We conclude that in addition to being regulated by intracellular Ca2+, Drosophila bestrophins are also novel members of the volume-regulated anion channel (VRAC) family that are necessary for cell volume homeostasis.

Lidocaine Promotes the Trafficking and Functional Expression of Nav1.8 Sodium Channels in Mammalian Cells

2007 ◽  
Vol 98 (1) ◽  
pp. 467-477 ◽  
Author(s):  
Juan Zhao ◽  
Rahima Ziane ◽  
Aurélien Chatelier ◽  
Michael E. O'Leary ◽  
Mohamed Chahine
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Biophysical Properties ◽  
Nociceptive Neurons ◽  
Mammalian Cell Lines ◽  
Na Currents

Nociceptive neurons of the dorsal root ganglion (DRG) express a combination of rapidly gating TTX-sensitive and slowly gating TTX-resistant Na currents, and the channels that produce these currents have been cloned. The Nav1.7 and Nav1.8 channels encode for the rapidly inactivating TTX-sensitive and slowly inactivating TTX-resistant Na currents, respectively. Although the Nav1.7 channel expresses well in cultured mammalian cell lines, attempts to express the Nav1.8 channel using similar approaches has been met with limited success. The inability to heterologously express Nav1.8 has hampered detailed characterization of the biophysical properties and pharmacology of these channels. In this study, we investigated the determinants of Nav1.8 expression in tsA201 cells, a transformed variant of HEK293 cells, using a combination of biochemistry, immunochemistry, and electrophysiology. Our data indicate that the unusually low expression levels of Nav1.8 in tsA201 cells results from a trafficking defect that traps the channel protein in the endoplasmic reticulum. Incubating the cultured cells with the local anesthetic lidocaine dramatically enhanced the cell surface expression of functional Nav1.8 channels. The biophysical properties of the heterologously expressed Nav1.8 channel are similar but not identical to those of the TTX-resistant Na current of native DRG neurons, recorded under similar conditions. Our data indicate that the lidocaine acts as a molecular chaperone that promotes efficient trafficking and increased cell surface expression of Nav1.8 channels.

scholarly journals Depletion of mitochondrial inorganic polyphosphate (polyP) in mammalian cells causes metabolic shift from oxidative phosphorylation to glycolysis.

2021 ◽  
Author(s):  
Maria E. Solesio ◽  
Lihan Xie ◽  
Brendan McIntyre ◽  
Mathew Ellenberger ◽  
Erna Mitaishvili ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Oxidative Phosphorylation ◽  
Mammalian Cells ◽  
High Energy ◽  
Hek293 Cells ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Metabolic Switch ◽  
Respiratory Parameters ◽  
Respiratory Chain Activity

Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical to those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.

Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture

2003 ◽  
Vol 284 (6) ◽  
pp. C1346-C1354 ◽  
Author(s):  
Oscar R. Colegio ◽  
Christina Van Itallie ◽  
Christoph Rahner ◽  
James Melvin Anderson
Keyword(s):  
Transmembrane Proteins ◽  
Extracellular Domain ◽  
Paracellular Permeability ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Ionic Selectivity ◽  
Extracellular Domains ◽  
Charge Selectivity ◽  
Darby Canine Kidney ◽  
Canine Kidney

Tight junctions (TJs) regulate paracellular permeability across epithelia and vary widely in their transepithelial electrical resistance (TER) and charge selectivity. The claudin family of transmembrane proteins influences these properties. We previously reported that claudin-4 increased TER ∼300% when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells and decreased the paracellular permeability for Na+ more than Cl− (Van Itallie C, Rahner C, and Anderson JM. J Clin Invest 107: 1319–1327, 2001). In comparison, we report here that expression of claudin-2 increases TER by only ∼20% and does not change the ionic selectivity of MDCK II cells from their cation-selective background. To test whether the extracellular domains of claudins-4 and -2 determine their unique paracellular properties, we determined the effects of interchanging these domains between claudins-4 and -2. Inducible expression of wild-type claudins and extracellular domain chimeras increased both the number and depth of fibrils, but the characteristic fibril morphologies of claudin-4 or -2 were not altered by switching extracellular domains. Like claudin-4, chimeras expressing the first or both extracellular domains of claudin-4 on claudin-2 increased TER severalfold and profoundly decreased the permeability of Na+ relative to Cl−. In contrast, chimeras expressing the first or both extracellular domains of claudin-2 on claudin-4 increased the TER by only ∼60 and ∼40%, respectively, and only modestly altered charge selectivity. These results support a model in which the claudins create paracellular channels and the first extracellular domain is sufficient to determine both paracellular charge selectivity and TER.

scholarly journals Ca2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl– Channels

2020 ◽  
Vol 8 ◽  
Author(s):  
Raquel Centeio ◽  
Jiraporn Ousingsawat ◽  
Rainer Schreiber ◽  
Karl Kunzelmann
Keyword(s):  
Endoplasmic Reticulum ◽  
Anion Channel ◽  
Time Dependent ◽  
Hek293 Cells ◽  
Cell Swelling ◽  
Bath Solution ◽  
Phospholipid Scramblase ◽  
Typical Time ◽  
Intracellular Ca ◽  
Cl Channels

All vertebrate cells activate Cl– currents (ICl,swell) when swollen by hypotonic bath solution. The volume-regulated anion channel VRAC has now been identified as LRRC8/SWELL1. However, apart from VRAC, the Ca2+-activated Cl– channel (CaCC) TMEM16A and the phospholipid scramblase and ion channel TMEM16F were suggested to contribute to cell swelling-activated whole-cell currents. Cell swelling was shown to induce Ca2+ release from the endoplasmic reticulum and to cause subsequent Ca2+ influx. It is suggested that TMEM16A/F support intracellular Ca2+ signaling and thus Ca2+-dependent activation of VRAC. In the present study, we tried to clarify the contribution of TMEM16A to ICl,swell. In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of ICl,swell by hypotonic bath solution (Hypo; 200 mosm/l) was Ca2+ dependent. TMEM16A augmented the activation of LRRC8A/C by enhancing swelling-induced local intracellular Ca2+ concentrations. In HT29 cells, knockdown of endogenous TMEM16A attenuated ICl,swell and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of ICl,swell by Hypo was attenuated by blocking receptors for inositol trisphosphate and ryanodine (IP3R; RyR), as well as by inhibiting Ca2+ influx. The data suggest that TMEM16A contributes directly to ICl,swell as it is activated through swelling-induced Ca2+ increase. As activation of VRAC is shown to be Ca2+-dependent, TMEM16A augments VRAC currents by facilitating Hypo-induced Ca2+ increase in submembraneous signaling compartments by means of ER tethering.

scholarly journals Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1391
Author(s):  
Andrey Kropotov ◽  
Veronika Kulikova ◽  
Kirill Nerinovski ◽  
Alexander Yakimov ◽  
Maria Svetlova ◽  
...  
Keyword(s):  
Nicotinic Acid ◽  
Mammalian Cells ◽  
Experimental Models ◽  
The Body ◽  
Nucleoside Transporters ◽  
Hek293 Cells ◽  
Nucleoside Transporter ◽  
Vitamin B3 ◽  
Animal Cells ◽  
Nicotinamide Riboside

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

scholarly journals Short Chain Fatty Acids Effect on Chloride Channel ClC-2 as a Possible Mechanism for Lubiprostone Intestinal Action

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1781
Author(s):  
Marcelo A. Catalán ◽  
Francisca Julio-Kalajzić ◽  
María Isabel Niemeyer ◽  
Luis Pablo Cid ◽  
Francisco V. Sepúlveda
Keyword(s):  
Fatty Acids ◽  
Mammalian Cells ◽  
Anion Channel ◽  
Synthetic Fatty Acid ◽  
Short Chain Fatty Acids ◽  
Physiological Effect ◽  
Short Chain ◽  
Distal Intestine ◽  
Dual Action ◽  
Chain Fatty Acids

Lubiprostone, a 20-carbon synthetic fatty acid used for the treatment of constipation, is thought to act through an action on Cl− channel ClC-2. Short chain fatty acids (SCFAs) are produced and absorbed in the distal intestine. We explore whether SCFAs affect ClC-2, re-examine a possible direct effect of lubiprostone on ClC-2, and use mice deficient in ClC-2 to stringently address the hypothesis that the epithelial effect of lubiprostone targets this anion channel. Patch-clamp whole cell recordings of ClC-2 expressed in mammalian cells are used to assay SCFA and lubiprostone effects. Using chamber measurements of ion current in mice deficient in ClC-2 or CFTR channels served to analyze the target of lubiprostone in the distal intestinal epithelium. Intracellular SCFAs had a dual action on ClC-2, partially inhibiting conduction but, importantly, facilitating the voltage activation of ClC-2. Intra- or extracellular lubiprostone had no effect on ClC-2 currents. Lubiprostone elicited a secretory current across colonic epithelia that was increased in mice deficient in ClC-2, consistent with the channel’s proposed proabsorptive function, but absent from those deficient in CFTR. Whilst SCFAs might exert a physiological effect on ClC-2 as part of their known proabsorptive effect, ClC-2 plays no part in the lubiprostone intestinal effect that appears mediated by CFTR activation.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

Sign in / Sign up

Export Citation Format

Share Document