A thiol-mediated active membrane transport of selenium by erythroid anion exchanger 1 protein.

In this paper, we describe a thiol-mediated and energy-dependent membrane transport of selenium by erythroid anion exchanger 1 (AE1, also known as band 3 protein). The AE1 is the most abundant integral protein of red cell membranes and plays a critical role in the carbon dioxide transport system in which carbon dioxide is carried as bicarbonate in the plasma. This protein mediates the membrane transport of selenium, an essential antioxidant micronutrient, from red cells to the plasma in a manner that is distinct from the already known anion exchange mechanism. In this pathway, selenium bound to the cysteine 93 of the hemoglobin ß chain (Hb-Cysß93) is transported by the relay mechanism to the Cys317 of the amino-terminal cytoplasmic domain of the AE1 on the basis of the intrinsic interaction between the two proteins and is subsequently exported to the plasma via the Cys843 of the membrane-spanning domain. The selenium export did not occur in plain isotonic buffer solutions and required thiols, such as albumin, in the outer medium. Such a membrane transport mechanism would also participate in the export pathways of the nitric oxide vasodilator activity and other thiol-reactive substances bound to the Hb-Cysß93 from red cells to the plasma and/or peripherals.

Thiol-dependent membrane transport of selenium through an integral protein of the red blood cell membrane.

The molecular details of the selenium metabolism and transport in living systems are still not completely understood, despite their physiological importance. Specifically, little is known about the membrane transport of selenium from most of the selenium containing compounds. In the present study, we investigated the mechanism for the membrane transport of selenium from red blood cells (RBCs) to the blood plasma. When the selenium distribution in the RBC ghost membrane after treatment with selenious acid was analyzed, nearly 70% of the selenium in the membrane was found to bind to the anion exchanger 1 (AE1) protein, which suggested that the integral protein AE1 is responsible for the membrane transport of selenium. The thiol dependency of the selenium export from the RBC to the blood plasma was examined using membrane permeable thiol reagents, i.e., N-ethylmaleimide (NEM) and tetrathionate (TTN). Treatment of the RBC with NEM, a thiol-alkylating reagent, resulted in modification of the thiol groups in the amino-terminal cytoplasmic domain (N-CPD) of the AE1, but not those in the membrane domain. Such an NEM treatment provided a marked inhibition of the selenium export from the RBC to the blood plasma. In addition, the treatment with TTN, a thiol-oxidizing reagent that forms intermolecular disulfide bonds, appeared to oxidize thiol groups in both the N-CPD and the membrane domain of AE1, which resulted in complete inhibition of the selenium export even during the initial period in which the export had a maximum velocity when using the thiol reagent-free treatment. Such complete inhibition of the selenium export from the TTN-treated RBC appeared to be due to the oligomerized AE1 proteins resulting from the intermolecularly formed disulfide bonds. These inhibitory effects using NEM and TTN suggested that thiol groups in the integral protein AE1 play essential roles in the membrane transport of the selenium from the RBCs to the blood plasma.

Molecular mapping of the conductance activity linked to tAE1 expressed in Xenopus oocyte.

It was previously shown that expressed in Xenopus oocyte the trout (tAE1) and the mouse (mAE1) anion exchangers behave differently: both elicit anion exchange activity but only tAE1 induces a transport of organic solutes correlated with an anion conductance. In order to identify the structural domains involved in the induction of tAE1 channel activity, chimeras have been prepared between mouse and trout AE1. As some constructs were not expressed at the plasma membrane, skate exchanger (skAE1) was used instead of mouse exchanger to complete the structure-function analysis. The present paper shows that skAE1, highly similar to mAE1, does not induce a chloride conductance when expressed in Xenopus oocyte. Construct expression analysis showed that only tAE1 transmembrane domain is linked to the anion conductance. More precisely, we identified two regions composed of helices 6, 7 and 8 and putative helices 12 and 13 which are required for this function.

Solution structure of a band 3 peptide inhibitor bound to aldolase: a proposed mechanism for regulating binding by tyrosine phosphorylation.

Human erythrocyte band 3 inhibits glycolytic enzymes, including aldolase, by binding these cytoplasmic enzymes at its N-terminus. Phosphorylation of Y8 disrupts inhibition, and there is evidence that in vivo glycolysis levels in erythrocytes are regulated in part by a phosphorylation/dephosphorylation signaling pathway. The structural basis for control by phosphorylation has been investigated by NMR studies on a complex between aldolase and a synthetic peptide corresponding to the first 15 residues of band 3 (MEELQDDYEDMMEEN-NH2). The structure of this band 3 peptide (B3P) when it is bound to rabbit muscle aldolase was determined using the exchange-transferred nuclear Overhauser effect (ETNOE). Two hundred NMR structures for B3P were generated by simulated annealing molecular dynamics with NMR-derived distance restraints and excluding electrostatic terms. Twenty structures were further refined against a force field including full partial charges. The important conformational feature of B3P in the bound state is a folded loop structure involving residues 4-9 and M12 that surrounds Y8 and is stabilized by a hydrophobic cluster with the ring of Y8 sandwiched between the methyl groups of L4 and M12. Differential line broadening indicates that this loop structure binds aldolase in a relatively specific manner, while terminal regions are structurally heterogeneous. To better understand B3P inhibition of aldolase and the mechanism of phosphorylation control, a complex was modeled by docking B3P into the active site of aldolase and optimizing the fit using restrained molecular dynamics and energy minimization. The B3P loop is complementary in conformation to the beta-barrel central core containing the aldolase active site residues. Binding is electrostatic in nature with numerous ionic and hydrogen-bonding interactions involving several conserved lysine and arginine residues of aldolase. How phosphorylation of band 3 could disrupt inhibition was considered by modeling a phosphoryl moiety onto Y8 of B3P. An energetic analysis with respect to rigid phosphate rotation suggests that aldolase inhibition is reversed primarily because of electrostatic repulsion between B3P residues that destabilizes the B3P loop formed in the complex. This proposed intramolecular mechanism for blocking protein--protein association by electrostatic repulsion with the phosphoryl group may be applicable to other protein--protein signaling complexes.

Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis.

To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3sMEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHIS in the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine subsitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors.

Anion transport function of mouse erythroid band 3 protein (AE1) does not require acylation of cysteine residue 861.

Cys-861 of mouse band 3 is equivalent to Cys-843 of human band 3, the only acylated cysteine residue in the anion exchanger AE1 of the red blood cell (Hamasaki et al. (1992) Progress Cell Res. 2, 65-71). Mutation of Cys-861 to serine or methionine caused no significant changes of band 3-mediated anion exchange as measured after expression of the appropriate cRNAs in Xenopus oocytes. Susceptibility to inhibition of transport by 4,4'-dinitrostilbene-2,2'-disulfonate and PCMBS was not affected. We conclude that palmitoylation is not an absolute requirement for the successful execution the anion transport function by the hydrophobic domain of band 3 in the plasma membrane.