Abstract
The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopus oocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [(14)C]methylamine (MA), had an apparent EC(50) of 1.6 mm and a maximum uptake rate (V(max)) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na(+) gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H(+) gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl- and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC(50) = 1.14 mm) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H(+)-coupled counter-transport mechanism.
Highlights
From the Departments of ‡Pathology and Laboratory Medicine and ¶Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104
Expressed Rh-associated glycoprotein (RhAG) was detected in oocyte membrane-enriched fractions as a single band (Fig. 1A), corresponding to the largest molecular weight band seen in native red blood cell membranes, suggesting that the expressed RhAG was fully glycosylated and properly trafficked to the plasma membrane
RhAG-mediated Uptake of [14C]MA—The characterization of ammonium ion transport is hindered by the lack of a convenient tracer for transport studies
Summary
CRNA Synthesis—Human RhAG and RhCE cDNAs were obtained by PCR from reticulocyte RNA. cDNAs were cloned into a pSP64 vector Oocyte Injection and [14C]Methylammonium Uptake Assay—Stage V and VI defolliculated oocytes were injected with 34 nl (1 ng/nl) of cRNA, or water for controls, and placed in individual wells in 96-well plates containing 200 l of SOS in mM (100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, pH 7.6, 200 mosM) with 2.5 mM sodium pyruvate, 100 g/ml gentamicin, and 100 M N-acetyl-Leu-Leu-norleucinal at 16 °C. Experiments were performed at room temperature by placing groups of six to eight oocytes in 200 l of low Kϩ (0.2 mM) SOS uptake buffer containing 1 Ci/ml [14C]methylammonium (MA) and various concentrations of unlabeled MA (20 M–50 mM) with or without competitors or inhibitors. Oocytes were preincubated for 25 min before uptake assay in buffers containing in mM 55 NaCl, 60 sodium acetate, 1.8 CaCl2, 1 MgCl2, 10 HEPES and adjusted to a final
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