Multiple epithelial Na+ channel domains participate in subunit assembly

2003 ◽  
Vol 285 (4) ◽  
pp. F600-F609 ◽  
Author(s):  
James B. Bruns ◽  
Baofeng Hu ◽  
Yoon J. Ahn ◽  
Shaohu Sheng ◽  
Rebecca P. Hughey ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Expression System ◽  
Functional Expression ◽  
Full Length ◽  
Na Channel ◽  
Xenopus Laevis Oocyte ◽  
Subunit Interactions ◽  
Α Subunit ◽  
Hydrophobic Domain ◽  
Membrane Spanning

Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC α-subunit that confer a dominant negative phenotype on functional expression of αβγ-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse αβγ-ENaC with either 1) the α-subunit first membrane-spanning domain and short downstream hydrophobic domain (α-M1H1); 2) α-M1H1 and its downstream hydrophilic extracellular loop (α-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; γ-GT) fused to the α-ECL (γ-GT-α-ECL); 4) the extracellular domain of a control type 1 transmembrane protein (Tac) fused to the α-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-α-H2M2); or 5) the α-subunit cytoplasmic COOH terminus (α-Ct) significantly reduced amiloride-sensitive Na+ currents in X. laevis oocytes. Functional expression of Na+ channels was not inhibited when full-length αβγ-ENaC was coexpressed with either 1) the α-ECL lacking a signal-anchor sequence, 2) α-M1H1 and α-Ct expressed as a fusion protein, 3) full-length γ-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either α-M1H1-ECL or α-Ct. Full-length FLAG-tagged α-, β-, or γ-ENaC coimmunoprecipitated with myc-tagged α-M1H1-ECL, whereas wild-type γ-GT did not. These data suggest that multiple sites within the α-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex.

Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits

1997 ◽  
Vol 273 (6) ◽  
pp. C1889-C1899 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Albert Tousson ◽  
Dale J. Benos
Keyword(s):  
Fluorescence Intensity ◽  
Expression System ◽  
Fold Increase ◽  
Na Channel ◽  
Β Subunit ◽  
Open Probability ◽  
Animal Pole ◽  
Α Subunit ◽  
Amino Acid Peptide ◽  
Xenopus Oocyte Expression

Using the Xenopus oocyte expression system, we examined the mechanisms by which the β- and γ-subunits of an epithelial Na+channel (ENaC) regulate α-subunit channel activity and the mechanisms by which β-subunit truncations cause ENaC activation. Expression of α-ENaC alone produced small amiloride-sensitive currents (−43 ± 10 nA, n = 7). These currents increased >30-fold with the coexpression of β- and γ-ENaC to −1,476 ± 254 nA ( n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the α-subunit of ENaC. Truncation of the last 75 amino acids of the β-subunit COOH terminus, as found in the original pedigree of individuals with Liddle’s syndrome, caused a 4.4-fold ( n = 17) increase of the amiloride-sensitive currents compared with wild-type αβγ-ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human β-ENaC significantly reduced the amiloride-sensitive currents by 40–50%. These observations suggest a tonic inhibitory role on the channel’s open probability ( P o) by the COOH terminus of β-ENaC. We conclude that the changes of current observed with coexpression of the β- and γ-subunits or those observed with β-subunit truncation are likely the result of changes of channel density in combination with large changes of P o.

scholarly journals Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its α subunit

1999 ◽  
Vol 104 (7) ◽  
pp. 967-974 ◽  
Author(s):  
Olivier Bonny ◽  
Ahmed Chraibi ◽  
Jan Loffing ◽  
Nicole Fowler Jaeger ◽  
Stefan Gründer ◽  
...  
Keyword(s):  
Functional Expression ◽  
Na Channel ◽  
Type I ◽  
Α Subunit ◽  
Epithelial Na Channel ◽  
Pseudohypoaldosteronism Type

scholarly journals Molecular cloning and functional expression of bovine spleen ecto-NAD+ glycohydrolase: structural identity with human CD38

1999 ◽  
Vol 345 (1) ◽  
pp. 43-52 ◽  
Author(s):  
Angélique AUGUSTIN ◽  
Hélène MULLER-STEFFNER ◽  
Francis SCHUBER
Keyword(s):  
Transmembrane Protein ◽  
Functional Expression ◽  
Full Length ◽  
Cysteine Residues ◽  
Sequence Information ◽  
Functional Domain ◽  
High Sequence Identity ◽  
Full Length Cdna ◽  
Nad Glycohydrolase ◽  
C Terminus

Bovine spleen ecto-NAD+ glycohydrolase, an archetypal member of the mammalian membrane-associated NAD(P)+ glycohydrolase enzyme family (EC 3.2.2.6), displays catalytic features similar to those of CD38, i.e. a protein originally described as a lymphocyte differentiation marker involved in the metabolism of cyclic ADP-ribose and signal transduction. Using amino acid sequence information obtained from NAD+ glycohydrolase and from a truncated and hydrosoluble form of the enzyme (hNADase) purified to homogeneity, a full-length cDNA clone was obtained. The deduced sequence indicates a protein of 278 residues with a molecular mass of 31.5 kDa. It predicts that bovine ecto-NAD+ glycohydrolase is a type II transmembrane protein, with a very short intracellular tail. The bulk of the enzyme, which is extracellular and contains two potential N-glycosylation sites, yields the fully catalytically active hNADase which is truncated by 71 residues. Transfection of HeLa cells with the full-length cDNA resulted in the expression of the expected NAD+ glycohydrolase, ADP-ribosyl cyclase and GDP-ribosyl cyclase activities at the surface of the cells. The bovine enzyme, which is the first ‘classical’ NAD(P)+ glycohydrolase whose structure has been established, presents a particularly high sequence identity with CD38, including the presence of 10 strictly conserved cysteine residues in the ectodomain and putative catalytic residues. However, it lacks two otherwise conserved cysteine residues near its C-terminus. Thus hNADase, the truncated protein of 207 amino acids, represents the smallest functional domain endowed with all the catalytic activities of CD38/NAD+ glycohydrolases so far identified. Altogether, our data strongly suggest that the cloned bovine spleen ecto-NAD+ glycohydrolase is the bovine equivalent of CD38.

scholarly journals Functional expression of the polymeric immunoglobulin receptor from cloned cDNA in fibroblasts.

1986 ◽  
Vol 102 (3) ◽  
pp. 911-919 ◽  
Author(s):  
D L Deitcher ◽  
M R Neutra ◽  
K E Mostov
Keyword(s):  
Transmembrane Protein ◽  
Expression System ◽  
Fibroblast Cell ◽  
Functional Expression ◽  
Secretory Component ◽  
Mouse Fibroblast ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Ligand Complex ◽  
Immunoglobulin Receptor

The polymeric immunoglobulin receptor, a transmembrane protein, is made by a variety of polarized epithelial cells. After synthesis, the receptor is sent to the basolateral surface where it binds polymeric IgA and IgM. The receptor-ligand complex is endocytosed, transported across the cell in vesicles, and re-exocytosed at the apical surface. At some point the receptor is proteolytically cleaved so that its extracellular ligand binding portion (known as secretory component) is severed from the membrane and released together with the polymeric immunoglobulin at the apical surface. We have used a cDNA clone coding for the rabbit receptor and a retroviral expression system to express the receptor in a nonpolarized mouse fibroblast cell line, psi 2, that normally does not synthesize the receptor. The receptor is glycosylated and sent to the cell surface. The cell cleaves the receptor to a group of polypeptides that are released into the medium and co-migrate with authentic rabbit secretory component. Cleavage and release of secretory component do not depend on the presence of ligand. The cells express on their surface 9,600 binding sites for the ligand, dimeric IgA. The ligand can be rapidly endocytosed and then re-exocytosed, all within approximately 10 min. Very little ligand is degraded. At least some of the ligand that is released from the cells is bound to secretory component. The results presented indicate that we have established a powerful new system for analyzing the complex steps in the transport of poly-Ig and the general problem of membrane protein sorting.

scholarly journals Functional Expression of Multidrug Resistance Protein 4 MRP4/ABCC4

2019 ◽  
Vol 24 (10) ◽  
pp. 1000-1008 ◽  
Author(s):  
David Hardy ◽  
Roslyn M. Bill ◽  
Anass Jawhari ◽  
Alice J. Rothnie
Keyword(s):  
Multidrug Resistance ◽  
Membrane Proteins ◽  
Transmembrane Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Functional Expression ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Multidrug Resistance Protein ◽  
Resistance Protein

To study the function and structure of membrane proteins, high quantities of pure and stable protein are needed. One of the first hurdles in accomplishing this is expression of the membrane protein at high levels and in a functional state. Membrane proteins are naturally expressed at low levels, so finding a suitable host for overexpression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein that is able to transport a range of organic anionic compounds (both endogenous and xenobiotic) out of the cell. This versatile transporter has been linked with extracellular signaling pathways and cellular protection, along with conferring drug resistance in cancers. Here we report the use of MRP4 as a case study to be expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Interestingly, using the baculovirus expression system with Sf9 insect cells produced the highest protein yields. Vesicular transport assays were used to confirm that MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabeled substrates. MRP4 transported fluo-cAMP in an ATP-dependent manner. The specificity of functional expression of MRP4 was validated by the use of nonhydrolyzable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilization and purification in order to better understand its function and structure.

scholarly journals Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron

2011 ◽  
Vol 301 (4) ◽  
pp. C862-C871 ◽  
Author(s):  
Jorge J. Pinilla-Tenas ◽  
Brian K. Sparkman ◽  
Ali Shawki ◽  
Anthony C. Illing ◽  
Colin J. Mitchell ◽  
...  
Keyword(s):  
Functional Properties ◽  
Cellular Uptake ◽  
Metal Ion ◽  
Transmembrane Protein ◽  
Expression System ◽  
Nitrilotriacetic Acid ◽  
Isolated Hepatocytes ◽  
Xenopus Laevis Oocyte ◽  
Ion Transporter ◽  
Broad Scope

Recent studies have shown that overexpression of the transmembrane protein Zrt- and Irt-like protein 14 (Zip14) stimulates the cellular uptake of zinc and nontransferrin-bound iron (NTBI). Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of 55Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe2+) over ferric ion (Fe3+). Zip14-mediated 55Fe2+ uptake was saturable ( K0.5 ≈ 2 μM), temperature-dependent (apparent activation energy, Ea = 15 kcal/mol), pH-sensitive, Ca2+-dependent, and inhibited by Co2+, Mn2+, and Zn2+. HCO3− stimulated 55Fe2+ transport. These properties are in close agreement with those of NTBI uptake in the perfused rat liver and in isolated hepatocytes reported in the literature. Zip14 also mediated the uptake of 109Cd2+, 54Mn2+, and 65Zn2+ but not 64Cu (I or II). 65Zn2+ uptake also was saturable ( K0.5 ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca2+ dependence of Zn2+ transport differed from those of Fe2+ transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.

3D Reconstruction of Na+, K+-ATpase from Tubular Crystals

2000 ◽  
Vol 6 (S2) ◽  
pp. 238-239
Author(s):  
W. J. Rice ◽  
H.S. Young ◽  
D.W. Martin ◽  
J. R. Sachs ◽  
D.L. Stokes
Keyword(s):  
Amino Acids ◽  
Transmembrane Protein ◽  
Phosphorylation Site ◽  
Animal Cells ◽  
Α Subunit ◽  
A Subunit ◽  
Extracellular Side ◽  
Membrane Spanning ◽  
P Type ◽  
Tubular Crystals

The Na+,K+-ATPase is a transmembrane protein, located in the plasma membrane of virtually all animal cells, which controls Na+ and K+ gradients. It is a member of the P-type ATPase family of ion pumps, a group of enzymes which pump ions against a concentration gradient, forming a phosphorylated intermediate during the pumping cycle. For each mole of ATP hydrolysed, 3 Na + ions are moved out of the cell and 2 K+ ions are moved into the cell. Unlike most other members of this family, which have one subunit, Na+, K+-ATPase is a heterodimer of α and β subunits. The a subunit consists of 1020 amino acids and has been predicted to have 10 membrane-spanning a-helices as well as a large cytoplasmic headpiece which forms the ATP binding and phosphorylation site. The α subunit, 300 amino acids in length, has one membrane spanning helix and has most of its mass located on the extracellular side of the membrane.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

scholarly journals Specific and Nonspecific Effects of Protein Kinase C on the Epithelial Na + Channel

2000 ◽  
Vol 115 (5) ◽  
pp. 559-570 ◽  
Author(s):  
Mouhamed S. Awayda
Keyword(s):  
Membrane Trafficking ◽  
Expression System ◽  
Na Channel ◽  
Specific Effects ◽  
Nonspecific Effects ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Baseline Values ◽  
Epithelial Na Channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na+ channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49–65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (τ) and a magnitude (ΔI V). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na+ channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35–45). Stimulation of PKC with 100 nM PMA decreased ΔIV in hENaC-expressing oocytes to a plateau at 57.1 ± 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (gNa) to 21.7 ± 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g Na was attributed to a decrease of membrane capacitance (C m), as well as the specific conductance (gm/Cm ). The effects on gm/Cm reached a plateau within 15 min, at ∼60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of Cm was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of Cm were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of gm and by changes of gm/Cm, indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.

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