Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits

2001 ◽  
Vol 281 (3) ◽  
pp. C982-C992 ◽  
Author(s):  
Craig Gatto ◽  
Scott M. McLoud ◽  
Jack H. Kaplan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Atpase Activity ◽  
Insect Cells ◽  
Expression System ◽  
Β Subunit ◽  
Α Subunit ◽  
Enzyme Preparations ◽  
High Five Cells

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.

scholarly journals Glycosylation is essential for biosynthesis of functional gastric H+, K+-ATPase in insect cells

1997 ◽  
Vol 321 (2) ◽  
pp. 419-424 ◽  
Author(s):  
Corné H. W. KLAASSEN ◽  
Jack A. M. FRANSEN ◽  
Herman G. P. SWARTS ◽  
Jan Joep H. H. M. De PONT
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Functional Properties ◽  
Insect Cells ◽  
Expression Level ◽  
Α Subunit ◽  
Atpase Subunits ◽  
Glycoprotein Processing ◽  
Α Subunits

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and 1-deoxymannojirimycin, inhibitors of glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated α-subunits and not of non-glycosylated α-subunits. Transport of the H+,K+-ATPase α-subunit to the plasma membrane but not its ability to assemble with the α-subunit depended on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

Colonic H-K-ATPase α- and β-subunits express ouabain-insensitive H-K-ATPase

2000 ◽  
Vol 278 (1) ◽  
pp. C182-C189 ◽  
Author(s):  
Pitchai Sangan ◽  
Sundararajah Thevananther ◽  
Sheela Sangan ◽  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Atpase Activity ◽  
Plasma Membranes ◽  
Expression System ◽  
Distal Colon ◽  
Rat Colon ◽  
Β Subunit ◽  
Α Subunit ◽  
Hek 293 ◽  
Transfected Cells ◽  
Rat Distal Colon

Active K absorption in the rat distal colon is energized by an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase α-subunit (HKcα) has been cloned and characterized (together with the β-subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive86Rb uptake. In contrast, HKcα, when expressed in Sf9 cells without a β-subunit, yielded evidence of ouabain-insensitive H-K-ATPase. Because a β-subunit (HKcβ) has recently been cloned from rat colon, this present study was initiated to determine whether H-K-ATPase and its sensitivity to ouabain are expressed when these two subunits (HKcα and HKcβ) are transfected into a mammalian cell expression system. Transfection of HEK-293 cells with HKcα and HKcβ cDNAs resulted in the expression of HKcα and HKcβ proteins and their delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was 1) saturable as a function of increasing K concentration with a K m for K of 0.63 mM; 2) inhibited by orthovanadate; and 3) insensitive to both ouabain and Sch-28080. In parallel transfection studies with HKcα and Na-K-ATPase β1 cDNAs and with HKcα cDNA alone, there was expression of ouabain-insensitive H-K-ATPase activity that was 60% and 21% of that in HKcα/HKcβ cDNA transfected cells, respectively. Ouabain-insensitive 86Rb uptake was also identified in cells transfected with HKcα and HKcβ cDNAs. These studies establish that HKcα cDNA with HKcβ cDNA express ouabain-insensitive H-K-ATPase similar to that identified in rat distal colon.

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.

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.

Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation

2003 ◽  
Vol 285 (4) ◽  
pp. C968-C976 ◽  
Author(s):  
O. Vagin ◽  
S. Denevich ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Glycosylation Site ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Hek 293 ◽  
Glycosylation Sites ◽  
293 Cells

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.

scholarly journals Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies.

1984 ◽  
Vol 98 (6) ◽  
pp. 2142-2147 ◽  
Author(s):  
P Quinn ◽  
G Griffiths ◽  
G Warren
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Semliki Forest Virus ◽  
Companion Paper ◽  
Intracellular Membranes ◽  
Surface Areas ◽  
Quantitative Immunoblotting ◽  
Total Membrane

Using two independent methods, incorporation of radioactive amino-acid and quantitative immunoblotting, we have determined that the rate of synthesis of each of the Semliki Forest virus (SFV) proteins in infected baby hamster kidney (BHK) cells is 1.2 X 10(5) copies/cell/min. Given the absolute surface areas of the endoplasmic reticulum and Golgi complex presented in the companion paper (Griffiths, G., G. Warren, P. Quinn , O. Mathieu - Costello , and A. Hoppeler , 1984, J. Cell Biol. 98:2133-2141), and the approximate time spent in these organelles during their passage to the plasma membrane (Green J., G. Griffiths, D. Louvard , P. Quinn , and G. Warren 1981, J. Mol. Biol. 152:663-698), the mean density of each viral protein in these organelles can be calculated to be 90 and 750 molecules/micron 2 membrane, respectively. In contrast, we have determined that the density of total endogenous integral membrane proteins in these organelles is approximately 30,000 molecules/micron 2 so that the spike proteins constitute only 0.28 and 2.3% of total membrane protein in the endoplasmic reticulum and Golgi, respectively. Quantitative immunoblotting was used to give direct estimates of the concentrations of one of the viral membrane protein precursors (E1) in subcellular fractions; these agreed closely with the calculated values. The data are discussed with respect to the sorting of transported proteins from those endogenous to the intracellular membranes.

scholarly journals Ivermectin as an inhibitor of the plasma membrane and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPases in rat vas deferens

2014 ◽  
Vol 23 (2) ◽  
pp. 75
Author(s):  
Humberto Muzi-Filho ◽  
Dominick Rodrigues Alves de Souza ◽  
Christianne Bretas Vieira Scaramello ◽  
Valeria Do Monti Nascimento Cunha
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Vas Deferens ◽  
Physiological Function ◽  
Binding Protein ◽  
Rat Vas Deferens ◽  
Release Channel ◽  
Fk506 Binding Protein ◽  
Fertility Status

ObjectiveThe present work investigated the effect of ivermectin on Ca2+ content and on the Ca2+-ATPase activity (represented by the plasma membrane Ca2+-ATPase and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase present in rat vas deferens.MethodsThe assays were carried out using ultracentrifuged homogenate preparations from rat vas deferens in the presence or absence of the 12-kDa FK506-binding protein-Ca2+ release channel complex. Measures of Ca2+ content and Ca2+ ATPase activity were then carried out in function of different concentrations of ivermectin. ResultsThe data show that ivermectin (10 μM) reduces the sarcoplasmic reticulum Ca2+ content in FK506-binding protein (+) and FK506-binding protein (-) fractions of ultracentrifuged homogenate from rat vas deferens (inhibition of 50% and 40%, respectively, p<0.05) and inhibits both the activities of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase and plasma membrane Ca2+-ATPases pumps (33% and 16%, respectively, p<0.05).ConclusionThese data suggest that ivermectin effects Ca2+ handling in the rat vas deferens, indicating that this drug could alter the contractility of this smooth muscle. Therefore, ivermectin could be an interesting pharmacological tool to alter the physiological function of vas deferens and to manipulate the fertility status of male rats.Indexing terms: Calcium. Ivermectin. Rats.

scholarly journals Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle

1985 ◽  
Vol 231 (3) ◽  
pp. 737-742 ◽  
Author(s):  
J Verbist ◽  
F Wuytack ◽  
L Raeymaekers ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Transport Atpase ◽  
Calmodulin Binding ◽  
Dependent Atpase ◽  
Inhibitory Antibodies ◽  
Stimulation Of

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.

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