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

The kinetics of sodium-stimulated phosphate flux and phosphate-stimulated s odium 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). Howeve r, 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-C 769), the molecular basis for the sodium-phosphate cotransporter in erythro cytes is unknown. Most cells express multiple isoforms, thus disallowing ex plication of isoform specific kinetics and function. We have found that ery throcyte membranes express one dominant isoform, hBNP-1, to which the kinet ics call thus be ascribed. In addition, because the erythrocyte Na-PO4 cotr ansporter can also mediate Li-PO4 cotransport, it has been suggested that t his transporter functions as the erythrocyte Na-Li exchanger whose activity is systematically altered in patients with bipolar disease and patients wi th essential hypertension. To determine the molecular basis for the sodium- phosphate cotransporter, we reasoned that if the kinetics of phosphate tran sport in a nucleated erythroid-like cell paralleled those of the Na-activat ed pathway in anucleated erythrocytes and yet were distinct, from those kno wn for other Na-PO4 cotransporters, then the expressed genes may be the sam e in both cell types. In this study, we show that the kinetics of sodium ph osphate cotransport were similar in anuclear human erythrocytes and K562 ce lls, a human erythroleukemic cell line. Although the erythrocyte fluxes wer e 750-fold smaller, the half-activation concentrations for phosphate and so dium and the relative cation specificities for activation of (PO4)-P-32 inf lux were similar. Na-activation curves for both cell types showed cooperati vity consistent with the reported stoichiometry of more than one Na cotrans ported per PO4. In K562 cells, external lithium activation of phosphate inf lux was also cooperative. Inhibition by arsenate, K-I = 2.6-2.7 mM, and rel ative inhibition by amiloride, amiloride analogs, phosphonoformate, and phl oretin were similar. These characteristics were different from those report ed for hNaPi-3 and hPiT-1 in other systems. PCR analysis of sodium-phosphat e cotransporter isoforms in K562 cells demonstrated the presence of mRNAs f or hPiT-1, hPiT-2, and hBNP-1. The mRNAs for hNaPi-10 and hNaPi-3, the othe r two known isoforms, were absent. Western analysis of erythrocytes and K56 2 cells with isoform-specific antibodies detected the presence of only hBNP -1, an isoform expressed in br-ain neurons and glia. The similarities in th e 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-phos phate cotransporter.