Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis

2011 ◽  
Vol 300 (4) ◽  
pp. F887-F897 ◽  
Author(s):  
Oded Edelheit ◽  
Israel Hanukoglu ◽  
Nathan Dascal ◽  
Aaron Hanukoglu
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
Epithelial Sodium Channels ◽  
Charged Residues ◽  
Inhibition Control ◽  
Two Parameters

Epithelial sodium channels (ENaC) are composed of three homologous subunits whose extracellular domains (ECD) form a funnel that directs ions from the lumen into the pore of ENaC. To examine the roles of conserved charged residues (Asp, Glu, Arg, and Lys) on ECD, we mutated 16 residues in human α-ENaC to alanine. The modified cRNAs were expressed in Xenopus laevis oocytes together with wild-type β- and γ-ENaC. The effect of each mutation was examined on three parameters: amiloride-sensitive Na+ conductance (assayed by the two-electrode voltage-clamp method), Na+-dependent self-inhibition of ENaC, and oocyte cell surface expression of ENaC (quantitated by confocal microscopy of yellow fluorescent protein linked to γ-ENaC). Mutation of 13 of 16 residues reduced the ENaC Na+ conductance (to 40–80% of WT). Mutation of only six residues showed a significant effect on the Na+ self-inhibition time constant (τ). All 16 mutants showed a strong correlation between ENaC activity and oocyte surface expression ( r = 0.62). Exclusion of four mutants showing the greatest effect on self-inhibition kinetics (Glu250 and Arg350 with τ = ∼30% of WT, and Asp393 and Glu530 with τ = ∼170% of WT) increased the correlation to r = 0.87. In the ASIC1 homotrimeric model, the homologs of α-ENaC Asp400 and Asp446 are exposed on the protein surface far from the other two chains. The mutations of these two residues showed the strongest effect on cell surface expression but had no effect on self-inhibition. Control mutations to a homologous charged residue (e.g., Asp to Glu) did not significantly affect ENaC activity. Changes in the two parameters, Na+ self-inhibition and oocyte surface expression level, accounted for the magnitude of reduction in ENaC activity as a result of the mutation to Ala. These results establish that while some conserved charged residues are part of the structure responsible for Na+ self-inhibition, most are essential for transport to the oocyte cell surface.

scholarly journals Abrogation of cell surface expression of human class I transplantation antigens by an adenovirus protein in Xenopus laevis oocytes.

1985 ◽  
Vol 101 (2) ◽  
pp. 540-547 ◽  
Author(s):  
L Severinsson ◽  
P A Peterson
Keyword(s):  
Cell Surface ◽  
Heavy Chain ◽  
Viral Protein ◽  
Surface Expression ◽  
Class I ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
Human Class ◽  
Heavy Chains ◽  
Transplantation Antigens

Class I transplantation antigens form complexes with a virus protein encoded in the early region E3 of the adenovirus-2 genome. The interaction between this viral glycoprotein, E19, and nascent human class I antigens has been examined by microinjecting purified mRNA into Xenopus laevis oocytes. Both E19 and the two class I antigen subunits, the heavy chain and beta 2-microglobulin (beta 2M), were efficiently translated. The heavy chains did not become terminally glycosylated, as monitored by endoglycosidase H digestion, and were not expressed on the oocyte surface unless they were associated with beta 2M. The E19 protein did not become terminally glycosylated, and we failed to detect this viral protein on the surface of the oocytes. Co-translation of heavy chain and E19 mRNA demonstrated that the two proteins associate intracellularly. However, neither protein appeared to be transported to the trans-Golgi compartment. Similar observations were made in adenovirus-infected HeLa cells. Heavy chains bound to beta 2M became terminally glycosylated in oocytes in the presence of low concentrations of E19. At high concentrations of the viral protein, no carbohydrate modifications and no cell surface expression of class I antigens were apparent. Thus, beta 2M and E19 have opposite effects on the intracellular transport of the heavy chains. These data suggest that adenovirus-2 may impede the cell surface expression of class I antigens to escape immune surveillance.

COMMD1 downregulates the epithelial sodium channel through Nedd4–2

2010 ◽  
Vol 298 (6) ◽  
pp. F1445-F1456 ◽  
Author(s):  
Ying Ke ◽  
A. Grant Butt ◽  
Marianne Swart ◽  
Yong Feng Liu ◽  
Fiona J. McDonald
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Sodium Channel ◽  
Rat Kidney ◽  
Copper Metabolism ◽  
Epithelial Sodium Channel ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes

The epithelial sodium channel (ENaC) is important for the long-term control of Na+ homeostasis and blood pressure. Our previous studies demonstrated that Copper Metabolism Murr1 Domain-containing protein 1 (COMMD1; previously known as Murr1), a protein involved in copper metabolism, inhibited amiloride-sensitive current in Xenopus laevis oocytes expressing ENaC ( J Biol Chem 279: 5429, 2004). In this study, we report that COMMD1 inhibits amiloride-sensitive current in mammalian epithelial cells expressing ENaC, that the COMM domain of COMMD1 is sufficient for this effect, and that knockdown of COMMD1 increases amiloride-sensitive current. COMMD1 is coexpressed with ENaC in rat kidney medulla cells. COMMD1 increased ubiquitin modification of ENaC and decreased its cell surface expression. COMMD1 abolished insulin-stimulated amiloride-sensitive current and attenuated the stimulation of current by activated serum and glucocorticoid-regulated kinase (SGK1). COMMD1 was found to interact with both SGK1 and Akt1/protein kinase B, and knockdown of COMMD1 enhanced the stimulatory effect of both SGK1 and Akt1 on amiloride-sensitive current. COMMD1's effects were reduced in the presence of ENaC proteins containing PY motif mutations, abolished in the presence of a dominant negative form of Nedd4–2, and knockdown of COMMD1 reduced the inhibitory effect of Nedd4–2 on ENaC, but did not enhance current when Nedd4–2 was knocked down. These data suggest that COMMD1 modulates Na+ transport in epithelial cells through regulation of ENaC cell surface expression and this effect is likely mediated via Nedd4–2.

scholarly journals SGK1 increases Na,K-ATP cell-surface expression and function in Xenopus laevis oocytes

2004 ◽  
Vol 448 (1) ◽  
pp. 29-35 ◽  
Author(s):  
Marija Zecevic ◽  
Dirk Heitzmann ◽  
Simone M. R. Camargo ◽  
Francois Verrey
Keyword(s):  
Cell Surface ◽  
Xenopus Laevis ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
And Function

scholarly journals Subunit Interactions Influence TSHR Multimerization

2010 ◽  
Vol 24 (10) ◽  
pp. 2009-2018 ◽  
Author(s):  
Rauf Latif ◽  
Krzysztof Michalek ◽  
Terry F. Davies
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Surface ◽  
Hormone Receptors ◽  
Fluorescent Protein ◽  
Surface Expression ◽  
Full Length ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Glycoprotein Hormone ◽  
Green Fluorescent

Abstract The TSH receptor (TSHR) is the key molecule influencing thyroid growth and development and is an antigenic target in autoimmune thyroid disease. The TSHR exists in monomeric and multimeric forms, and it has been shown previously that multimeric complexes of the TSHR preferentially localize in lipid rafts. However, unlike other glycoprotein hormone receptors, the TSHR exists in several forms on the cell membrane due to intramolecular cleavage of its ectodomain, which causes the production of α- and β-subunits of various lengths. After cleavage and reduction of disulfide bonds, α-subunits consisting of the receptor ectodomain may be lost from the cell surface by receptor shedding, leading to accumulation of excess β-subunits within the membrane. Because cell surface expression of these various forms of the TSHR is critical to receptor signaling and autoimmune responses, we set out to model the influence of β-subunits on full-length TSHRs. To study this interaction, we generated three truncated ectodomain β-subunits linked to green fluorescent protein (named β-316, -366, and -409) as examples of native cleaved forms of the TSHR. These constructs were transfected into human embryonic kidney 293 cells in the presence and absence of the full-length receptor. Whereas the β-316 and β-366 forms showed cell surface expression, the expression of β-409 was primarily intracellular. Cotransfection of the β-subunits with a full-length hemagglutinin-tagged wild-type (WT) receptor (HT-WT-TSHR) in both transient and stable systems caused a significant decrease in surface expression of the full-length WT receptors. This decrease was not seen with control plasmid consisting of a plasma membrane-targeted protein tagged to red fluorescent protein. To ascertain if this response was due to homointeraction of the truncated β-constructs with the WT-TSHRs, we immunoprecipitated membranes prepared from the cotransfected cells using antihemagglutinin and then probed with anti-green fluorescent protein. These studies confirmed dimerization of the β-subunits with the WT full-length receptor, and this interaction was further observed in vivo by fluorescence resonance energy transfer. We then studied the functional consequences of this interaction on TSHR signaling by examining Gαs-mediated signals. The well-expressed truncated constructs, when coexpressed with full-length TSHR, did not alter constitutive cAMP levels, but there was a significant decrease in TSH-induced cAMP generation. Furthermore, we observed that truncated β-316 and β-366 had faster internalization rate, which may lead to a significant decrease in the expression of the full-length receptor on the cell surface, thus contributing to the decreased signaling response. However, the decrease in surface receptors may also be due to inhibition of newly formed receptors reaching the surface as result of receptor-receptor interaction. It is well known that under normal physiological conditions both cleaved and uncleaved TSHR forms coexist on the cell surface of normal thyrocytes. Our studies allow us to conclude, therefore, that multimerization of cleaved/ truncated forms of the β-subunits with the full-length TSHR has a profound influence on TSHR internalization and signaling. Hence, the degree of intramolecular cleavage must also modulate TSHR signaling.

Persistent HIV Transcription Induces Sialyl-Lewis-X Glyco-Antigen Cell-Surface Expression

2020 ◽  
Author(s):  
Florent Colomb ◽  
Leila B. Giron ◽  
Leticia Kuri Cervantes ◽  
Tongcui Ma ◽  
Samson Adeniji ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Sialyl Lewis X ◽  
Cell Surface Expression ◽  
Lewis X ◽  
Hiv Transcription ◽  
Sialyl Lewis

Influence of β-D-mannuronic acid, as a new member of non-steroidal anti-inflammatory drugs family, on expression pattern of chemokines and their receptors in rheumatoid arthritis

2019 ◽  
Vol 16 ◽  
Author(s):  
Mona Aslani ◽  
Arman Ahmadzadeh ◽  
Zahra Aghazadeh ◽  
Majid Zaki-Dizaji ◽  
Laleh Sharifi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mrna Expression ◽  
Surface Expression ◽  
Phase Iii ◽  
Cell Surface Expression ◽  
Optimum Dose ◽  
High Dose ◽  
Mannuronic Acid ◽  
Anti Inflammatory ◽  
High Doses

Background: : Based on the encouraging results of phase III clinical trial of β-D-mannuronic acid (M2000) (as a new anti-inflammatory drug) in patients with RA, in this study, we aimed to evaluate the effects of this drug on the expression of chemokines and their receptors in PBMCs of RA patients. Methods:: PBMCs of RA patients and healthy controls were separated and the patients' cells were treated with low, moderate and high doses (5, 25 and 50 μg/mL) of M2000 and optimum dose (1 μg/mL) of diclofenac, as a control in RPMI-1640 medium. Real-time PCR was used for evaluating the mRNA expression of CXCR3, CXCR4, CCR2, CCR5 and CCL2/MCP-1. Cell surface expression of CCR2 was investigated using flow cytometry. Results:: CCR5 mRNA expression reduced significantly, after treatment of the patients' cells with all three doses of M2000 and optimum dose of diclofenac. CXCR3 mRNA expression down-regulated significantly followed by treatment of these cells with moderate and high doses of M2000 and optimum dose of diclofenac. CXCR4 mRNA expression declined significantly after treatment of these cells with moderate and high doses of M2000. CCL2 mRNA expression significantly reduced only followed by treatment of these cells with high dose of M2000, whereas, mRNA and cell surface expressions of CCR2 diminished significantly followed by treatment of these cells with high dose of M2000 and optimum dose of diclofenac. Conclusion:: According to our results, M2000 through the down-regulation of chemokines and their receptors may restrict the infiltration of immune cells into the synovium.

Sign in / Sign up

Export Citation Format

Share Document