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The molecular basis for Na-dependent phosphate transport in human erythrocytes and K562 cells.

Timmer RT, Gunn RB - J. Gen. Physiol. (2000)

Bottom Line: We have found that erythrocyte membranes express one dominant isoform, hBNP-1, to which the kinetics can thus be ascribed.Western analysis of erythrocytes and K562 cells with isoform-specific antibodies detected the presence of only hBNP-1, an isoform expressed in brain neurons and glia.The similarities in the kinetics and the expression of only hBNP-1 protein in the two cell types is strong evidence that hBNP-1 is the erythrocyte and K562 cell sodium-phosphate cotransporter.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

ABSTRACT
The kinetics of sodium-stimulated phosphate flux and phosphate-stimulated sodium flux in human red cells have been previously described (Shoemaker, D.G., C.A. Bender, and R.B. Gunn. 1988. J. Gen. Physiol. 92:449-474). However, despite the identification of multiple isoforms in three gene families (Timmer, R.T., and R.B. Gunn. 1998. Am. J. Physiol. Cell Physiol. 274:C757-C769), the molecular basis for the sodium-phosphate cotransporter in erythrocytes is unknown. Most cells express multiple isoforms, thus disallowing explication of isoform-specific kinetics and function. We have found that erythrocyte membranes express one dominant isoform, hBNP-1, to which the kinetics can thus be ascribed. In addition, because the erythrocyte Na-PO(4) cotransporter can also mediate Li-PO(4) cotransport, it has been suggested that this transporter functions as the erythrocyte Na-Li exchanger whose activity is systematically altered in patients with bipolar disease and patients with essential hypertension. To determine the molecular basis for the sodium-phosphate cotransporter, we reasoned that if the kinetics of phosphate transport in a nucleated erythroid-like cell paralleled those of the Na-activated pathway in anucleated erythrocytes and yet were distinct from those known for other Na-PO(4) cotransporters, then the expressed genes may be the same in both cell types. In this study, we show that the kinetics of sodium phosphate cotransport were similar in anuclear human erythrocytes and K562 cells, a human erythroleukemic cell line. Although the erythrocyte fluxes were 750-fold smaller, the half-activation concentrations for phosphate and sodium and the relative cation specificities for activation of (32)PO(4) influx were similar. Na-activation curves for both cell types showed cooperativity consistent with the reported stoichiometry of more than one Na cotransported per PO(4). In K562 cells, external lithium activation of phosphate influx was also cooperative. Inhibition by arsenate, K(I) = 2.6-2.7 mM, and relative inhibition by amiloride, amiloride analogs, phosphonoformate, and phloretin were similar. These characteristics were different from those reported for hNaPi-3 and hPiT-1 in other systems. PCR analysis of sodium-phosphate cotransporter isoforms in K562 cells demonstrated the presence of mRNAs for hPiT-1, hPiT-2, and hBNP-1. The mRNAs for hNaPi-10 and hNaPi-3, the other two known isoforms, were absent. Western analysis of erythrocytes and K562 cells with isoform-specific antibodies detected the presence of only hBNP-1, an isoform expressed in brain neurons and glia. The similarities in the kinetics and the expression of only hBNP-1 protein in the two cell types is strong evidence that hBNP-1 is the erythrocyte and K562 cell sodium-phosphate cotransporter.

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Activation of 32PO4 influx by monovalent cations in erythrocytes and K562 cells. Monovalent cation activation of 32PO4 influx in the presence of 1.0 mM external phosphate, pH 7.4, 37ºC, and fluxes were carried out as described with each indicated cation present at 143 mM using the chloride salt. The standard errors of six determinations in one of four similar experiments.
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Figure 4: Activation of 32PO4 influx by monovalent cations in erythrocytes and K562 cells. Monovalent cation activation of 32PO4 influx in the presence of 1.0 mM external phosphate, pH 7.4, 37ºC, and fluxes were carried out as described with each indicated cation present at 143 mM using the chloride salt. The standard errors of six determinations in one of four similar experiments.

Mentions: Activation of the 32PO4 influx by monovalent cations is shown in Fig. 4 for K562 cells. Chloride media with the only monovalent cation being 143 mM N-methyl-d-glucamine, choline, rubidium, potassium, or cesium gave small or negligible 32PO4 influxes into K562 cells. Only lithium and sodium were able to activate the flux relative to the other cations. The activation of the 32PO4 influx in K562 cells (Fig. 4) by 143 mM external lithium was 19% (±6%, n = 15) of the activation by 143 mM external sodium (100%). This relative activation by these two alkali cations was the same in erythrocytes (S. Elmariah and R.B. Gunn, manuscript in preparation) and was much greater than the relative activation (<2%) reported in HEK-293 cells expressing hNaPi-3 (Timmer and Gunn 1998) or seen in oocytes expressing hPiT-1 (Olah et al. 1994).


The molecular basis for Na-dependent phosphate transport in human erythrocytes and K562 cells.

Timmer RT, Gunn RB - J. Gen. Physiol. (2000)

Activation of 32PO4 influx by monovalent cations in erythrocytes and K562 cells. Monovalent cation activation of 32PO4 influx in the presence of 1.0 mM external phosphate, pH 7.4, 37ºC, and fluxes were carried out as described with each indicated cation present at 143 mM using the chloride salt. The standard errors of six determinations in one of four similar experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233690&req=5

Figure 4: Activation of 32PO4 influx by monovalent cations in erythrocytes and K562 cells. Monovalent cation activation of 32PO4 influx in the presence of 1.0 mM external phosphate, pH 7.4, 37ºC, and fluxes were carried out as described with each indicated cation present at 143 mM using the chloride salt. The standard errors of six determinations in one of four similar experiments.
Mentions: Activation of the 32PO4 influx by monovalent cations is shown in Fig. 4 for K562 cells. Chloride media with the only monovalent cation being 143 mM N-methyl-d-glucamine, choline, rubidium, potassium, or cesium gave small or negligible 32PO4 influxes into K562 cells. Only lithium and sodium were able to activate the flux relative to the other cations. The activation of the 32PO4 influx in K562 cells (Fig. 4) by 143 mM external lithium was 19% (±6%, n = 15) of the activation by 143 mM external sodium (100%). This relative activation by these two alkali cations was the same in erythrocytes (S. Elmariah and R.B. Gunn, manuscript in preparation) and was much greater than the relative activation (<2%) reported in HEK-293 cells expressing hNaPi-3 (Timmer and Gunn 1998) or seen in oocytes expressing hPiT-1 (Olah et al. 1994).

Bottom Line: We have found that erythrocyte membranes express one dominant isoform, hBNP-1, to which the kinetics can thus be ascribed.Western analysis of erythrocytes and K562 cells with isoform-specific antibodies detected the presence of only hBNP-1, an isoform expressed in brain neurons and glia.The similarities in the kinetics and the expression of only hBNP-1 protein in the two cell types is strong evidence that hBNP-1 is the erythrocyte and K562 cell sodium-phosphate cotransporter.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

ABSTRACT
The kinetics of sodium-stimulated phosphate flux and phosphate-stimulated sodium flux in human red cells have been previously described (Shoemaker, D.G., C.A. Bender, and R.B. Gunn. 1988. J. Gen. Physiol. 92:449-474). However, despite the identification of multiple isoforms in three gene families (Timmer, R.T., and R.B. Gunn. 1998. Am. J. Physiol. Cell Physiol. 274:C757-C769), the molecular basis for the sodium-phosphate cotransporter in erythrocytes is unknown. Most cells express multiple isoforms, thus disallowing explication of isoform-specific kinetics and function. We have found that erythrocyte membranes express one dominant isoform, hBNP-1, to which the kinetics can thus be ascribed. In addition, because the erythrocyte Na-PO(4) cotransporter can also mediate Li-PO(4) cotransport, it has been suggested that this transporter functions as the erythrocyte Na-Li exchanger whose activity is systematically altered in patients with bipolar disease and patients with essential hypertension. To determine the molecular basis for the sodium-phosphate cotransporter, we reasoned that if the kinetics of phosphate transport in a nucleated erythroid-like cell paralleled those of the Na-activated pathway in anucleated erythrocytes and yet were distinct from those known for other Na-PO(4) cotransporters, then the expressed genes may be the same in both cell types. In this study, we show that the kinetics of sodium phosphate cotransport were similar in anuclear human erythrocytes and K562 cells, a human erythroleukemic cell line. Although the erythrocyte fluxes were 750-fold smaller, the half-activation concentrations for phosphate and sodium and the relative cation specificities for activation of (32)PO(4) influx were similar. Na-activation curves for both cell types showed cooperativity consistent with the reported stoichiometry of more than one Na cotransported per PO(4). In K562 cells, external lithium activation of phosphate influx was also cooperative. Inhibition by arsenate, K(I) = 2.6-2.7 mM, and relative inhibition by amiloride, amiloride analogs, phosphonoformate, and phloretin were similar. These characteristics were different from those reported for hNaPi-3 and hPiT-1 in other systems. PCR analysis of sodium-phosphate cotransporter isoforms in K562 cells demonstrated the presence of mRNAs for hPiT-1, hPiT-2, and hBNP-1. The mRNAs for hNaPi-10 and hNaPi-3, the other two known isoforms, were absent. Western analysis of erythrocytes and K562 cells with isoform-specific antibodies detected the presence of only hBNP-1, an isoform expressed in brain neurons and glia. The similarities in the kinetics and the expression of only hBNP-1 protein in the two cell types is strong evidence that hBNP-1 is the erythrocyte and K562 cell sodium-phosphate cotransporter.

Show MeSH
Related in: MedlinePlus