The molecular basis for Na-dependent phosphate transport in human erythrocytes and K562 cells.
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.
Timmer
R T
RT
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Gunn
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000097:blood* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D020014:K562 Cells; D007700:Kinetics; D008969:Molecular Sequence Data; D010710:Phosphates / Q000097:blood*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000097:blood / Q000235:genetics; D012964:Sodium / Q000097:blood*; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
D002352:Carrier Proteins; D017931:DNA Primers; D010710:Phosphates; D020033:Protein Isoforms; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D012964:Sodium