scholarly journals Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion

1997 ◽  
Vol 138 (6) ◽  
pp. 1333-1341 ◽  
Author(s):  
Christof Zitt ◽  
Alexander G. Obukhov ◽  
Carsten Strübing ◽  
Andrea Zobel ◽  
Frank Kalkbrenner ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Receptors ◽  
Calcium Stores ◽  
Channel Activity ◽  
Ovary Cells ◽  
Cytosolic Side ◽  
Green Fluorescent ◽  
Inside Out

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.

Hands-on experiments on glycemia regulation and type 1 diabetes

2015 ◽  
Vol 39 (3) ◽  
pp. 232-239 ◽  
Author(s):  
M. Mingueneau ◽  
A. Chaix ◽  
N. Scotti ◽  
J. Chaix ◽  
A. Reynders ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Green Fluorescent Protein ◽  
Chinese Hamster Ovary ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Pancreatic Cells ◽  
Green Fluorescent

In the present article, we describe a 3-day experimental workshop on glycemia regulation and type 1 diabetes that engages students in open-ended investigations and guided experiments leading to results that are not already known to them. After an initial questioning phase during which students observe PowerPoint slides depicting the glycemia (blood glucose levels) of individuals in various situations, students design, execute, and interpret experiments to address one of the following questions: 1) Which criteria must an animal model of diabetes fulfill? 2) How do pancreatic cells maintain glycemia constant? and 3) Is there a way to produce an insulin protein similar to the one released by human pancreatic cells? Students then 1) measure glycemia and glycosuria in control mice and in a mouse model of type 1 diabetes (Alloxan-treated mice), 2) measure the release of insulin by pancreatic β-cells (INS-1 cell line) in response to different concentrations of glucose in the extracellular medium, and 3) transfect Chinese hamster ovary cells with a plasmid coding for green fluorescent protein, observe green fluorescent protein fluorescence of some of the transfected Chinese hamster ovary cells under the microscope, and observe the characteristics of human insulin protein and its three-dimensional conformation using RASMOL software. At the end of the experimental session, students make posters and present their work to researchers. Back at school, they may also present their work to their colleagues.

scholarly journals A Sperm-Associated WD Repeat Protein Orthologous to Chlamydomonas PF20 Associates with Spag6, the Mammalian Orthologue of Chlamydomonas PF16

2002 ◽  
Vol 22 (22) ◽  
pp. 7993-8004 ◽  
Author(s):  
Zhibing Zhang ◽  
Rossana Sapiro ◽  
David Kapfhamer ◽  
Maja Bucan ◽  
Jeff Bray ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chromosome 1 ◽  
Chromosome 2 ◽  
Flagellar Motility ◽  
Protein Fusion ◽  
Ovary Cells ◽  
Central Apparatus ◽  
Green Fluorescent

ABSTRACT cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.

Functional polymorphisms in the α-subunit of the human epithelial Na+ channel increase activity

2006 ◽  
Vol 290 (4) ◽  
pp. F821-F827 ◽  
Author(s):  
Qiusheng Tong ◽  
Anil G. Menon ◽  
James D. Stockand
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Severe Form ◽  
Na Channel ◽  
Channel Activity ◽  
Wild Type ◽  
Α Subunit ◽  
Ovary Cells ◽  
Functional Polymorphisms ◽  
Black Populations

Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Gain-of-function mutations in ENaC cause Liddle's syndrome: a severe form of inheritable hypertension. Several polymorphisms in α-hENaC possibly associated with abnormal Na+ handling by the kidney and the salt-sensitive hypertension prevalent in black populations have been reported. The functional effects of α-hENaC polymorphisms on channel activity, however, remain controversial and have not been directly tested in a mammalian background. We ask here whether polymorphisms at positions 334, 618, and 663 in α-hENaC influence channel activity. Activity of wild-type (A334, C618, A663) and polymorphic ENaC expressed in Chinese hamster ovary cells was assessed with patch-clamp electrophysiology. While the A334T polymorphism had little effect on macroscopic ENaC currents, the C618F and A663T polymorphisms significantly increased ENaC activity >3.3- and 1.6-fold, respectively. Similarly, polymorphic ENaC had greater activity compared with wild-type channels in excised patches with activity of C618F and A663T channels increased 3.8- and 2.6-fold, respectively. Unitary channel conductances and reversal potentials were not different for polymorphic and wild-type ENaC. Increases in activity resulted primarily from increases in the apparent number of active (polymorphic) channels in the plasma membrane. Moreover, addition of a reducing agent to the cytosol significantly increased activity of wild-type ENaC equal to that of C618F polymorphic channels but had no effect on these latter channels. These results are consistent with the C618F and A663T polymorphisms leading to elevated ENaC activity with the possibility that they facilitate altered Na+ handling by the kidney.

Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation

2004 ◽  
Vol 287 (3) ◽  
pp. C737-C745 ◽  
Author(s):  
Horia Vais ◽  
Rugang Zhang ◽  
William W. Reenstra
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Channel Activity ◽  
Ovary Cells ◽  
Channel Opening ◽  
Excised Patches ◽  
A Site ◽  
R Domain

To better understand the mechanisms by which PKA-dependent phosphorylation regulates CFTR channel activity, we have assayed open probabilities ( Po), mean open time, and mean closed time for a series of CFTR constructs with mutations at PKA phosphorylation sites in the regulatory (R) domain. Forskolin-stimulated channel activity was recorded in cell-attached and inside-out excised patches from transiently transfected Chinese hamster ovary cells. Wild-type CFTR and constructs with a single Ser-to-Ala mutation as well as octa (Ser-to-Ala mutations at 8 sites) and constructs with one or two Ala-to-Ser mutations were studied. In cell-attached patches, Ser-to-Ala mutations at amino acids 700, 795, and 813 decreased Po, whereas Ser-to-Ala mutations at 737 and 768 increased Po. In general, differences in Po were due to differences in mean closed time. For selected constructs with either high or low values of Po, channel activity was measured in excised patches. With 1 mM ATP, Po was similar to that observed in cell-attached patches, but with 10 mM ATP, all constructs tested showed elevated Po values. ATP-dependent increases in Po were due to reductions in mean closed time. These results indicate that R-domain phosphorylation affects ATP binding and not the subsequent steps of hydrolysis and channel opening. A model was developed whereby R-domain phosphorylation, in a site-dependent manner, alters equilibrium between forms of CFTR with low and high affinities for ATP.

scholarly journals A single-wavelength flow cytometric approach using redox-sensitive green fluorescent protein probes for measuring redox stress in live cells

BioTechniques ◽  
2021 ◽  
Author(s):  
Elizabeth R Denn ◽  
Joseph M Schober
Keyword(s):  
Oxidative Stress ◽  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Recovery Phase ◽  
Live Cells ◽  
Redox Stress ◽  
Green Fluorescent

Cellular redox changes are common in apoptosis, immune function, signaling pathways and cancer. The authors aimed to develop a single-wavelength method using the superior fluorescence sensitivity of a flow cytometer for measuring redox-sensitive green fluorescent protein signal during oxidative stress in cell lines. The single-wavelength method was able to discern small differences in oxidative stress between cell lines and between the cytoplasmic and mitochondrial compartments within the same cell line. In Chinese hamster ovary cells, the mitochondrial matrix compartment was more sensitive to oxidative stress compared with MDA-MB-231 cells, and the rapid changes in redox state were followed by a slow recovery phase. The authors conclude that this simplified method is useful and preferred for studies where alterations in overall redox-sensitive green fluorescent protein expression are controlled.

scholarly journals CFTR Gating I

2005 ◽  
Vol 125 (4) ◽  
pp. 361-375 ◽  
Author(s):  
Silvia G. Bompadre ◽  
Tomohiko Ai ◽  
Jeong Han Cho ◽  
Xiaohui Wang ◽  
Yoshiro Sohma ◽  
...  
Keyword(s):  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Kinetic Mechanism ◽  
Inhibitory Effect ◽  
Unexpected Result ◽  
Ovary Cells ◽  
Gating Mechanisms ◽  
Inside Out ◽  
R Domain

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed ΔR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the ΔR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal ΔR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of ΔR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and ΔR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the ΔR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

A glycosylated antitumor ether lipid kills cells via paraptosis-like cell death

2009 ◽  
Vol 87 (2) ◽  
pp. 401-414 ◽  
Author(s):  
Pranati Samadder ◽  
Robert Bittman ◽  
Hoe-Sup Byun ◽  
Gilbert Arthur
Keyword(s):  
Cell Death ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ether Lipid ◽  
Protein Fusion ◽  
Protein Marker ◽  
Anticancer Properties ◽  
Lc3 Puncta ◽  
Green Fluorescent

Glycosylated antitumor ether lipids (GAELs) have superior anticancer properties relative to the alkyllysophospholipid class, but there have been no studies of the mechanisms of these compounds. The prototype GAEL, 1-O-hexadecyl-2-O-methyl-3-O-(2′-amino-2′-deoxy-β-d-glucopyranosyl)-sn-glycerol (Gln), effectively killed mouse embryonic fibroblasts (MEFs) lacking key molecules involved in caspase-dependent apoptosis, and cell death was not prevented by caspase inhibitors. Gln did not cause a loss of mitochondrial membrane potential, even in rounded-up dying cells. Gln stimulated the appearance and accumulation of LC3-II, a protein marker for autophagy, in a variety of cells, including wild-type MEFs, but not in MEFs lacking ATG5, a key protein required for autophagy. Gln induced LC3 puncta formation in Chinese hamster ovary cells stably expressing a LC3–green fluorescent protein fusion protein. Thus, Gln appears to induce autophagy. Autophagy was mTOR-independent and was not inhibited by 3-methyladenine or wortmannin. Although Gln is toxic, cellular ability to undergo autophagy was not essential for its toxicity. Furthermore, the GAEL analog 2-deoxy-C-Glc induced LC3 puncta formation but did not kill the cells. Gln, but not 2-deoxy-C-Glc, caused the accumulation of cytoplasmic acidic vacuoles in the cells. Our data suggest that GAELs may activate autophagy; however, GAELs do not kill cells by apoptosis or autophagy but rather by a paraptosis-like cell death mechanism.

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