• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.181-181
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• 1996

The (Na, K) ATPase is a membrane ion transporting ATPase composed of an ${\alpha}$ catalytic subunit and a ${\beta}$ glycoprotein subunit. The topology of the rat ${\alpha}$1 and ${\beta}$1 subunits has been studied by insertion of epitope(s) : at the NH2-terminus and COOH-terminus and between Glu117 and Glul18, Lys828 and Arg829, Gln900 and Trp901, and Va1939 and Phe940 of the ${\alpha}$ subunit; and at the NH2-terminus and COOH-terminus and between Glu228 and Tyr229 of the ${\beta}$ subunit. The epitope-tagged ${\alpha}$l, constructs were expressed in HeLa cells to select for stable cell lines expressing a functional (Na, K)ATPase. All constructs, except for the one tagged between Gln900 and Trp901, resulted in ouabain-resistant colonies indicating that modified proteins retained functional integrity. The epitope-tagged ${\beta}$ constructs were transiently expressed in Cos-7 cells. The orientation of the epitopes with respect to the cell membrane was revealed by indirect immunofluorescence performed on permeabilized and non-permeabilized cells expressing the (Na, K)ATPase chains. The results indicate that the ${\alpha}$ subunit has 4 transmembrane segments in the COOH terminal membrane bound domain between residues 760 and 938, and that both the NH2-terminus and the COOH-terminus are in the cytosol; it was not determined whether there are more transmembrane segments between residue 938 and the COOH-terminus. The ${\beta}$ subunit has only one transmembrane spanning region with the NH2-terminus in the cytosol and the COOH-terminus on the extracytoplasmic surface of the plasma membrane.

• BMB Reports
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• v.54 no.3
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• pp.164-169
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• 2021

Neuronal growth regulator 1 (NEGR1) is a GPI-anchored membrane protein that is involved in neural cell adhesion and communication. Multiple genome wide association studies have found that NEGR1 is a generic risk factor for multiple human diseases, including obesity, autism, and depression. Recently, we reported that Negr1-/- mice showed a highly increased fat mass and affective behavior. In the present study, we identified Na/K-ATPase, beta1-subunit (ATP1B1) as an NEGR1 binding partner by yeast two-hybrid screening. NEGR1 and ATP1B1 were found to form a relatively stable complex in cells, at least partially co-localizing in membrane lipid rafts. We found that NEGR1 binds with ATP1B1 at its C-terminus, away from the binding site for the alpha subunit, and may contribute to intercellular interactions. Collectively, we report ATP1B1 as a novel NEGR1-interacting protein, which may help deciphering molecular networks underlying NEGR1-associated human diseases.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.260-266
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• 2005

IgE-dependent histamine releasing factor (HRF), previously known as P23/P21 or translationally controlled tumor protein (TCTP), induces the degranulation of histamine in mast cell and basophil. Yeast two hybrid results showed that HRF interacts with the alpha subunit of Na, K-ATPase, suggesting that HRF is a regulator for governing the activity of Na, K-ATPase. In this study, we examined the interaction of HRF and Wa,K-ATPase after treatments of various PKC isotype inhibitors. Membrane fractionation, pull-down assay and immunoprecipitation results showed that PKC $\alpha,\;PKC\;\beta,\;\delta$ subunits are involved in the phosphorylation of HRF. However, these results did not correlate with the results of histamine release assay since histamine release assay results suggested that some PKC isotype inhibitors induced the histamine release in RBL-2H3 cell.

• The Korean Journal of Physiology
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• v.19 no.1
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• pp.15-23
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• 1985

Previous biochemical studies indicate that $(Na^++K^+)-ATPase$ is composed of two subunits, ${\alpha}$ and ${\beta}$, in a form of ${\alpha}_2{\beta}_2$ with a molecular weight of approximately 300,000 daltons. There is also suggestive evidence that the $Na^+$, $K^+$ pump in human erythrocytes occurs in a complex with some glycolytic enzymes. We assessed here in situ assembly size of the $(Na^++K^+)-ATPase$ of human erythrocytes by applying classical target theory to radiation inactivation data of the ouabain-sensitive sodium flux and ATP hydrolysis of intact cells and ghosts. Cells(in the presence of cryoprotective agent) and ghosts were irradiated at $-45^{\circ}C$ to $-50^{\circ}C$ with an increasing dose of a 1.5 MeV electron beam, and after thawing, the pump and/or enzyme activities were assayed. Each activity measured was decreased as a simple exponential function of radiation dose, from which a radiation sensitive volume (target size) was calculated. When intact cells were used, the target size of both $(Na^++K^+)-ATPase$ and $Na^+$, $K^+$ pump was found to be approximately 600,000 daltons. This target size of the ATPase was reduced to approximately 325,000 daltons if the cells were pretreated with strophanthidin. When ghosts were used, the target size of the ATPase was again approximately 325,000 daltons. Our target size measurement suggests that, in intact cells, the $(Na^++K^+)-ATPase/Na^+,K^+$ pump exists either as a dimer of $(\alpha\beta)_2$ which is a functional unit or as a monomer of $(\alpha\beta)_2$ but in tight complex with other enzyme or enzymes. The results also suggest that this dimeric or heterocomplex association is dissociated during ghost preparation and strophanthidin treatment.

• The Korean Journal of Physiology
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• v.30 no.2
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• pp.257-267
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• 1996

Diabetes mellitus is associated with a wide range of pathophysiologic changes in the kidney. This study was designed to examine the mechanisms by which glucose modulates the expression of polarized membrane transport functions in primary cultured rabbit renal proximal tubule cells. Results are as follows: The rate of 30 minute $Rb^{+}$ uptake was significantly higher($137.76{\pm}5.40%$) in primary renal tubular cell cultures treated with 20 mM glucose than that of 5 mM glucose. Not the level of mRNA for the ${\alpha}$ subunit of Na, K-ATPase but that of ${\beta}$ subunit was elevated in primary cultures treated with high glucose. The initial rate of methyl-${\alpha}$-D-glucopyranoside(${\alpha}$-MG) uptake was significantly lower($71.91{\pm}3.02%$) in monolayers treated with 20 mM glucose than that of 5 mM glucose. There was a tendency of an increase in phlorizin binding site in cells treated with 5 mM glucose. However, 3-O-methyl-D-glucose(3-O-MG) uptake was not affected by glucose concentration in culture media. TPA inhibited $Rb^{+}$ uptake by $63.61{\pm}1.94\;and\;45.80{\pm}1.36%$ and ${\alpha}$-MG uptake by $48.54{\pm}3.69\;and\;41.87{\pm}6.70%$ in the cells treated with 5 and 20 mM glucose, respectively. Also TPA inhibited mRNA expression of Na/glucose cotransporter in cells grown in 5mM glucose medium. cAMP significantly stimulated ${\alpha}$-MG uptake by $114.65{\pm}5.70%$ in cells treated with 5mM glucose, while it did not affect ${\alpha}$-MG uptake in cell treated with 20 mM glucose. However, cAMP inhibited $Rb^{+}$ uptake by $76.69{\pm}4.16\;and\;66.87{\pm}2.41%$ in cells treated with 5 and 20 mM glucose, respectively. In conclusion, the activity of the renal proximal tubular Na,K-ATPase is elevated in high glucose concentration. In contrast, the activity of the Na/glucose cotransport system is inhibited. High glucose may in part affect the activity of the Na,K-ATPase and the Na/glucose cotransport system by controlling the protein kinase C and/or A signal transduction pathway in primary cultured renal proximal tubule cells.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.31 no.1
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• pp.24-30
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• 2005

The flow of saliva is controlled entirely by nervous stimuli. The present study was aimed to explore the role of sympathetic and parasympathetic nerves in the regulation of sodium transporters and water channels in the salivary gland. Rats were denervated of their sympathetic and parasympathetic nerves to the submandibular gland, and the expression of sodium transporters and water channels was determined. The expression of either ${\alpha}-1$ or ${\beta}-1$ subunit of Na, K-ATPase was not significantly affected by the sympathetic denervation. On the contrary, the expression of both subunits was decreased by the parasympathetic denervation. The expression of ${\alpha}-,\;{\beta}-$, and ${\gamma}$-subunits of ENaC was not significantly affected by the sympathetic denervation, but was increased by the parasympathetic denervation. On the contrary, the expression of NHE3 was markedly decreased by both the sympathetic and the parasympathetic denervation. The sympathetic denervation significantly increased the expression of AQP1, while the parasympathetic denervation was without effect. The sympathetic and parasympathetic denervation significantly increased the expression of AQP4. The sympathetic denervation did not affect the expression of AQP5, but the parasympathetic denervation significantly decreased it. These results suggest that sympathetic and parasympathetic nerves have tonic effects on the regulation of sodium transporters and AQP water channels in the salivary gland. The sympathetic and parasympathetic denervation may then result in alterations of secretory rate and electrolyte composition of the saliva.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.1
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• pp.1-7
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• 2006

The present study was aimed to explore the role of sympathetic and parasympathetic nerves in the regulation of sodium transporters and water channels in the salivary gland. Rats were denervated of their sympathetic and parasympathetic nerves to the submandibular gland, and the glandular expression of sodium transporters and water channels was determined by Western blot analysis. The expression of either ${\alpha}1$ or ${\beta}1$ subunit of Na, K-ATPase was not significantly affected either by the sympathetic or by the parasympathetic denervation. The expression of subunits of epithelial sodium channels was significantly increased both in the denervated and contralateral glands either by the sympathetic or by the parasympathetic denervation. Neither the sympathetic nor the parasympathetic denervation significantly altered the expression of aquaporin-1 (AQP1). Nor was the expression of AQP4 affected significantly by the parasympathetic or the sympathetic denervation. On the contrary, the expression of AQP5 was significantly increased not only by the parasympathetic but also by the sympathetic denervation. These results suggest that sympathetic and parasympathetic nerves have tonic regulatory effects on the regulation of certain sodium transporters and AQP water channels in the salivary gland.