Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial

Summary In this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that 32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2–6×10⁶ sec^–1 at 22°C is calculated suggesting a channel mechanism.We would like to thank Kate Van Fossen for her faithful technical support. This work was supported by NIH grants No. HL-20985 and HL-37593.     

Molecular Mechanisms of Urea Transport

Physiologic data provided evidence for specific urea transporter proteins in red blood cells and kidney inner medulla. During the past decade, molecular approaches resulted in the cloning of several urea transporter cDNA isoforms derived from two gene families: UT-A and UT-B. Polyclonal antibodies were generated to the cloned urea transporter proteins, and their use in integrative animal studies resulted in several novel findings, including: (1) UT-B is the Kidd blood group antigen; (2) UT-B is also expressed in many non-renal tissues and endothelial cells; (3) vasopressin increases UT-A1 phosphorylation in rat inner medullary collecting duct; (4) the surprising finding that UT-A1 protein abundance and urea transport are increased in the inner medulla during conditions in which urine concentrating ability is reduced; and (5) UT-A protein abundance is increased in uremia in both liver and heart. This review will summarize the knowledge gained from studying molecular mechanisms of urea transport and from integrative studies into urea transporter protein regulation.     

A novel mutation at the JK locus causing Jknull phenotype in a Chinese family

Jk (kidd) blood group antigens are carried by the urea transporter UT-B[1,2]. The Jknull phenotype, lack-ing urea permeability in erythrocytes[3,4], has a very low frequency in all populations except Polynesians and Finns[5]. In Japan, only 14 individuals with Jk (a-b-) phenotype were identified from 638460 screened donor’s blood samples using the 2 mol/L urea solution hemolysis test[6]. The frequency of Jknull is 0.27% in Polynesian, about 0.03% in Finland[7], and extremely rare in Fran…     

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbations of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.     

Target analysis studies of red cell water and urea transport

Radiation inactivation was used to determine the nature and molecular weight of water and urea transporters in the human red cell. Red cells were frozen to -50 degrees C in a cryoprotectant solution, irradiated with 1.5 MeV electrons, thawed, washed and assayed for osmotic water and urea permeability by stopped-flow light scattering. The freezing and thawing process did not affect the rates of water or urea transport or the inhibitory potency of p-chloromercuribenzenesulfonate (pCMBS) on water transport and of phloretin on urea transport. Red cell urea transport inactivated with radiation (0-4 Mrad) with a single target size of 469 +/- 36 kDa. 40 microM phloretin inhibited urea flux by approx. 50% at each radiation dose, indicating that urea transporters surviving radiation were inhibitable. Water transport did not inactivate with radiation; however, the inhibitory potency of 2.5 mM pCMBS decreased from 86 +/- 1% to 4 +/- 9% over a 0-2 Mrad dose range. These studies suggest that red cell water transport either required one or more low-molecular-weight proteins, or is lipid-mediated, and that the pCMBS-binding site which regulates water flow inactivates with radiation. These results also suggest that red cell urea transport is mediated by a specific, high-molecular-weight protein. These results do not support the hypothesis that a band 3 dimer (190 kDa) mediates red cell osmotic water and urea transport.     

UT-B1 urea transporter plays a noble role as active water transporter in C6 glial cells

Since our experimental results suggest that UT-B1 functions as active water transporter against osmotic gradient in C6 glial cells, we report here for the first time the evidence for the active water transport. Exposure of C6 cells to a hyperosmotic solution containing glycerol or sucrose produced cell shrinkage due to water efflux according to osmotic gradient for water movement. On the other hand, C6 cells show cell swelling against osmotic gradient for water movement just after exposure to a hyperosmotic solution containing urea, indicating that water influx against osmotic gradient for water movement is accelerated by urea; i.e., urea performs active water transport. A specific inhibitor of UT-B, pCMBS, blocked the urea-induced swelling. The urea-induced cell swelling was significantly suppressed in the siRNA-induced UT-B1-knockdown C6 cells. Taken together, these observations indicate that UT-B1 acts as an active water transporter, providing a new model on active water transport.     

Effect of pCMBS on anion transport in human red cell membranes.

The kinetics of binding of the mercurial sulfhydryl reagent, pCMBS (p-chloromercuribenzene sulfonate), to the extracellular site(s) at which pCMBS inhibits water and urea transport across the human red cell membrane, have previously been characterized. To determine whether pCMBS binding alters Cl- transport, we measured Cl-/NO3- exchange by fluorescence enhancement, using the dye SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium). An essentially instantaneous extracellular phase of pCMBS inhibition is followed by a much slower intracellular phase, correlated with pCMBS permeation. We attribute the instantaneous phase to competitive inhibition of Cl- binding to band 3 by the pCMBS anion. The ID50 of 2.0 +/- 0.1 mM agrees with other organic sulfonates, but is very much greater than that of pCMBS inhibition of urea and water transport, showing that pCMBS reaction with water and urea transport inhibition sites has no effect on anion exchange. The intracellular inhibition by 1 mM pCMBS (1 h) is apparently non-competitive with Ki = 5.5 +/- 6.3 mM, presumably an allosteric effect of pCMBS binding to an intracellular band 3-related sulfhydryl group. After N-ethylmaleimide (NEM) treatment to block these band 3 sulfhydryl groups, there is apparent non-competitive inhibition with Ki = 2.1 +/- 1.2 mM, which suggests that pCMBS reacts with one of the NEM-insensitive sulfhydryl groups on a protein that links band 3 to the cytoskeleton, perhaps ankyrin or bands 4.1 and 4.2.     

Control of red cell urea and water permeability by sulfhydryl reagents

The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.     

Polymorphism of SLC14A1 encoding human erythrocytic Kidd antigens in the Chinese population

The SLC14A1 gene, which encodes the important Kidd blood group antigens, has not been systematically?analyzed at the molecular level in Chinese individuals. In this study, SLC14A1 genetic polymorphism was examined in Chinese individuals with Jk(a+b-), Jk(a+b+), and Jk(a-b+) expression. The Kidd phenotype was determined for 146 specimens using monoclonal anti-Jk^a and -Jk^b antibodies. From these, 87 specimens were Jk(a-b+), 21 were Jk(a+b-), and 38 were Jk(a+b+). According to the Kidd phenotype results, 20 specimens were randomly selected from each group, i.e., Jk(a-b+), Jk(a+b-), and Jk(a+b+), for the molecular analyses of exons 3 to 11 of the SLC14A1 gene. Novel alleles were detected in the SLC14A1 gene, including IVS3-106A, IVS3-99A, exon3 130G, IVS4-299G, IVS4-293G, IVS4+211C, IVS4 +230C, exon6 499A, exon6 588A, IVS7-68T, IVS9+244G, and IVS10-153T, indicating that the locus harbored significant polymorphism. We also showed that IVS4-299, IVS7-68, and IVS10-153 were novel SNPs absolutely associated with exon 8 nt. 838. The minor allele frequencies were all greater than 10% and all SNPs in the Chinese population showed Vel antigen expression on RBC membranes. We identified 12 SNPs in the SLC14A1 gene in the Chinese population, IVS3-106A, IVS3-99A, exon3 130G, IVS4-299G, IVS4-293G, IVS4+211C, IVS4 +230C, exon6 499A, exon6 588A, IVS7-68T, IVS9+244G, and IVS10-153T. Our results also indicated that three novel SNPs produced Jk^a and Jk^b antigens in Chinese individuals.     

The frequencies of Kidd blood group antigens and phenotypes among Saudi blood donors in Southwestern Saudi Arabia

BackgroundThe patients who require transfusion are prevalent in the Jazan Province, Saudi Arabia. Therefore, it is essential to know the frequency of blood group antigens in such a population. The Kidd blood group system (JK) has two antithetical antigens, Jk^a and Jk^b. Antibodies to these antigens may result in delayed hemolytic transfusion reactions. The present study investigated the frequencies of Jk^a and Jk^b and the phenotypes among Saudi blood donors living in the Jazan Province.MethodsOne hundred and forty-three samples from anonymous Saudi volunteer blood donors in the Jazan Province were serotype to detect Jk^a and Jk^b using gel card technology and determine the phenotypes of the JK blood group system.ResultsThe prevalence of Jk^a and Jk^b antigens were 90.64% (n = 126) and 69.40% (n = 93), respectively. The JK phenotypes were 34.96% Jk(a + b ? ) (n = 51), 12.59% Jk(a ? b + ) (n = 18), 52.45% Jk(a + b + ) (n = 75), and 0% Jk(a ? b ? ). The frequencies of the JK phenotypes in the Jazan population were significantly different from those in the Asian population (P < 0.05).ConclusionsWe reported the frequencies of the Jk^a and Jk^b antigens and the distribution of the JK phenotypes in a group of Saudi blood donors in the Jazan Province, Saudi Arabia. The phenotype Jk(a + b + ) was the most common among the study population. Furthermore, this study emphasizes the significance of identifying the frequency of JK antigens and phenotypes in the provinces of Saudi Arabia.     

Chemical modification of the neutral amino acid transport system L of Chinese hamster ovary cells with p-chloromercuribenzene sulfonate.

Branched-chain and aromatic neutral amino acids enter mammalian cells predominantly through a Na(+)-independent transport agency called System L. The sulfhydryl specific reagent p-chloromercuribenzene sulfonate (pCMBS) has been shown to be a potent inactivator of System L transport activity in Chinese hamster ovary cells, however, inactivation by pCMBS can be prevented by the presence of System L-specific substrate amino acids during the inactivation reaction. In addition, the presence of amino acids that are not substrates for System L have no effect on pCMBS inactivation of System L. Inactivation of System L activity by pCMBS was sensitive to pH and reversible by incubation with dithiothreitol. These findings suggest that there is a sulfhydryl group in, or very near, the amino acid-binding site of the System L transporter of CHO cells. Substrate protection, however, could be explained by conformational changes in the transporter associated with substrate binding. The presence of a substrate protectable sulfhydryl group on the System L transporter would aid in the attempt to identify this transporter using the technique of differential labeling.     

Relation between red cell anion exchange and urea transport

The new distilbene compound, DCMBT (4,4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385-389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.     

Relation between red cell anion exchange and urea transport

The new distilbene compound, DCMBT (4,4′-dichloromercuric-2,2,2′,2′-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385–389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.     

Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues

A new polyclonal antibody to the humanerythrocyte urea transporter UT-B detects a broad band between 45 and65 kDa in human erythrocytes and between 37 and 51 kDa in raterythrocytes. In human erythrocytes, the UT-B protein is the Kidd (Jk)antigen, and Jk(a+b+) erythrocytes express the 45- to 65-kDa band.However, in Jk null erythrocytes [Jk(ab)], only a faint band at55 kDa is detected. In kidney medulla, a broad band between 41 and 54 kDa, as well as a larger band at 98 kDa, is detected. Human and ratkidney show UT-B staining in nonfenestrated endothelial cells indescending vasa recta. UT-B protein and mRNA are detected in rat brain,colon, heart, liver, lung, and testis. When kidney medulla or liverproteins are analyzed with the use of a native gel, only a singleprotein band is detected. UT-B protein is detected in cultured bovineendothelial cells. We conclude that UT-B protein is expressed in morerat tissues than previously reported, as well as in erythrocytes.

     

The differential role of Cys-421 and Cys-429 of the Glut1 glucose transporter in transport inhibition by p-chloromercuribenzenesulfonic acid (pCMBS) or cytochalasin B (CB).

Cys-421 and Cys-429 of Glut1 were replaced by site-directed mutagenesis in order to investigate their involvement in basal glucose transport and transport inhibition. Neither of the two cysteine residues was essential for basal 2-deoxy-D-glucose uptake in Xenopus oocytes expressing the respective mutant M421 and M429. If applied from the external side, the poorly permeable sulfhydryl-reactive agent pCMBS inhibited 2-deoxy-D-glucose uptake of Glut1- and M421-expressing Xenopus oocytes but failed to affect uptake of the Cys-429 mutant. This is in agreement with the proposed two-dimensional model of Glut1 confirming that Cys-429 is the only residue exposed to the surface of the plasma membrane. The replacement of Cys-421 at the exofacial end of helix eleven caused a partial protection of 3-O-methylglucose transport inhibition by CB; this residue may thus be involved in stabilizing an adjacent local tertiary structure necessary for the full activity of this inhibitor.     

Kinetic independence between red cell anion exchange and urea transport

Urea equilibrium exchange fluxes were measured in human red cells under conditions which recruit the anion transporter into an outward-facing or an inward-facing state (with respect to the anion transport site). Regardless of these conditions, urea transport always occurred at the same rate: 41 +/- 2 mol.(kg cell solids.min)-1 with 1.5 M urea at 0 degrees C. These data suggest that the pathway on the band-3 protein which mediates anion transport is kinetically uncoupled from urea transport and is probably not involved in the transport of urea across the red cell membrane.     

Blood groups in Native Americans: a look beyond ABO and Rh

The study presents comparisons between blood group frequencies beyond ABO and Rh blood systems in Native American populations and previously published data from Brazilian blood donors. The frequencies of Diego (c.2561C>T, rs2285644), Kell (c.578C>T, rs8176058), Duffy (c.125A>G, rs12075, c.1−67T>C, rs2814778) and Kidd (c.838A>G, rs1058396) variants in Kaingang (n=72) and Guarani (n=234) populations from Brazil (1990-2000) were obtained and compared with data from these populations sampled during the 1960s and with individuals of different Brazilian regions. Data showed high frequencies of DI*01 and FY*01 alleles: 11.8% and 57.6% in Kaingang and 6.8% and 75.7% in Guarani groups, respectively. The main results indicated: (1) reduction in genetic distance over time of Kaingang and Guarani in relation to other Brazilian populations is suggestive of ongoing admixture; (2) significant differences in some frequencies of blood group markers (especially Diego, Kidd and Duffy) in relation to Native Americans and individuals from different geographical regions of Brazil. Our study shows that the frequency of red blood cell polymorphisms in two Native American groups is very different from that of blood donors, when we evaluated blood groups different from ABO and Rh systems, suggesting that a better ethnic characterization of blood unit receptors is necessary.