Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells

1997 ◽  
Vol 434 (5) ◽  
pp. 632-638 ◽  
Author(s):  
Dominique Trouet ◽  
Bernd Nilius ◽  
Thomas Voets ◽  
G. Droogmans ◽  
J. Eggermont
Keyword(s):  
Ion Channels ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Gfp Expression

Integration and integrity of a bovine papillomavirus expression vector in different mammalian cells

1987 ◽  
Vol 6 (2) ◽  
pp. 91-105 ◽  
Author(s):  
M. Ruohonen-Lehto ◽  
K. Korpela ◽  
H. Söderlund ◽  
I. Ulmanen
Keyword(s):  
Mammalian Cells ◽  
Expression Vector ◽  
Bovine Papillomavirus

scholarly journals Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽  
2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Zackary Tajin Park
Keyword(s):  
Phage Display ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Restriction Enzymes ◽  
Phage Display Library ◽  
Single Chain ◽  
Phage Display Technology ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4).  LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

scholarly journals Hydrogen, Bicarbonate, and Their Associated Exchangers in Cell Volume Regulation

2021 ◽  
Vol 9 ◽  
Author(s):  
Yizeng Li ◽  
Xiaohan Zhou ◽  
Sean X. Sun
Keyword(s):  
Ion Channels ◽  
Cell Volume ◽  
Volume Regulation ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Water Flux ◽  
Cell Volume Regulation ◽  
Sodium Potassium ◽  
Before And After

Cells lacking a stiff cell wall, e.g., mammalian cells, must actively regulate their volume to maintain proper cell function. On the time scale that protein production is negligible, water flow in and out of the cell determines the cell volume variation. Water flux follows hydraulic and osmotic gradients; the latter is generated by various ion channels, transporters, and pumps in the cell membrane. Compared to the widely studied roles of sodium, potassium, and chloride in cell volume regulation, the effects of proton and bicarbonate are less understood. In this work, we use mathematical models to analyze how proton and bicarbonate, combined with sodium, potassium, chloride, and buffer species, regulate cell volume upon inhibition of ion channels, transporters, and pumps. The model includes several common, widely expressed ion transporters and focuses on obtaining generic outcomes. Results show that the intracellular osmolarity remains almost constant before and after cell volume change. The steady-state cell volume does not depend on water permeability. In addition, to ensure the stability of cell volume and ion concentrations, cells need to develop redundant mechanisms to maintain homeostasis, i.e., multiple ion channels or transporters are involved in the flux of the same ion species. These results provide insights for molecular mechanisms of cell volume regulation with additional implications for water-driven cell migration.

Delivering Ion Channels to Mammalian Cells by Membrane Fusion

2003 ◽  
pp. 275-296
Author(s):  
David C. Johns ◽  
Uta C. Hoppe ◽  
Eduardo Marbán ◽  
Brian O'Rourke
Keyword(s):  
Ion Channels ◽  
Membrane Fusion ◽  
Mammalian Cells

scholarly journals A Drosophila ribosomal protein functions in mammalian cells.

1990 ◽  
Vol 10 (9) ◽  
pp. 4524-4528 ◽  
Author(s):  
C G Maki ◽  
D D Rhoads ◽  
J J Diaz ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Resistance Mutation ◽  
Chinese Hamster ◽  
Ribosomal Subunits ◽  
Cdna Expression ◽  
Protein Functions

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

scholarly journals PIEZO ion channel is required for root mechanotransduction in Arabidopsis thaliana

2020 ◽  
Author(s):  
Seyed A. R. Mousavi ◽  
Adrienne E Dubin ◽  
Wei-Zheng Zeng ◽  
Adam M. Coombs ◽  
Khai Do ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Ion Channels ◽  
Ion Channel ◽  
Mammalian Cells ◽  
Plant Root ◽  
Mechanical Strain ◽  
Root Tip ◽  
Root Tips ◽  
Mechanosensitive Ion Channels ◽  
Mechanical Constraints

SummaryPlant roots adapt to the mechanical constraints of the soil to grow and absorb water and nutrients. As in animal species, mechanosensitive ion channels in plants are proposed to transduce external mechanical forces into biological signals. However, the identity of these plant root ion channels remains unknown. Here, we show that Arabidopsis thaliana PIEZO (AtPIEZO) has preserved the function of its animal relatives and acts as an ion channel. We present evidence that plant PIEZO is highly expressed in the columella and lateral root cap cells of the root tip which experience robust mechanical strain during root growth. Deleting PIEZO from the whole plant significantly reduced the ability of its roots to penetrate denser barriers compared to wild type plants. piezo mutant root tips exhibited diminished calcium transients in response to mechanical stimulation, supporting a role of AtPIEZO in root mechanotransduction. Finally, a chimeric PIEZO channel that includes the C-terminal half of AtPIEZO containing the putative pore region was functional and mechanosensitive when expressed in naive mammalian cells. Collectively, our data suggest that Arabidopsis PIEZO plays an important role in root mechanotransduction and establishes PIEZOs as physiologically relevant mechanosensitive ion channels across animal and plant kingdoms.

scholarly journals Hydrogen sulfide upregulates acid-sensing ion channels via the MAPK-Erk1/2 signaling pathway

Function ◽  
2021 ◽  
Author(s):  
Zhong Peng ◽  
Stephan Kellenberger
Keyword(s):  
Hydrogen Sulfide ◽  
Ion Channels ◽  
Signaling Pathway ◽  
Cortical Neurons ◽  
Mammalian Cells ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Membrane Expression ◽  
Concentration Dependent Manner ◽  
Acid Sensing Ion Channels

Abstract Hydrogen sulfide (H2S) emerged recently as a new gasotransmitter and was shown to exert cellular effects by interacting with proteins, among them many ion channels. Acid-sensing ion channels (ASICs) are neuronal voltage-insensitive Na+ channels activated by extracellular protons. ASICs are involved in many physiological and pathological processes, such as fear conditioning, pain sensation and seizures. We characterize here the regulation of ASICs by H2S. In transfected mammalian cells, the H2S donor NaHS increased the acid-induced ASIC1a peak currents in a time- and concentration-dependent manner. Similarly, NaHS potentiated also the acid-induced currents of ASIC1b, ASIC2a and ASIC3. An upregulation induced by the H2S donors NaHS and GYY4137 was also observed with the endogenous ASIC currents of cultured hypothalamus neurons. In parallel with the effect on function, the total and plasma membrane expression of ASIC1a was increased by GYY4137, as determined in cultured cortical neurons. H2S also enhanced the phosphorylation of extracellular signal-regulated kinase, which belongs to the family of mitogen-activated protein kinases (MAPKs). Pharmacological blockade of the MAPK signaling pathway prevented the GYY4137-induced increase of ASIC function and expression, indicating that this pathway is required for ASIC regulation by H2S. Our study demonstrates that H2S regulates ASIC expression and function, and identifies the involved signaling mechanism. Since H2S shares several roles with ASICs, as e.g. facilitation of learning and memory, protection during seizure activity and modulation of nociception, it may be possible that H2S exerts some of these effects via a regulation of ASIC function.

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