Reversible Band 3-Hemoglobin Association Serves as a Molecular Switch for Oxygen-Dependent Regulation of Red Blood Cell Properties

Human erythrocytes experience large variations in oxygen tension during their repeated transit between the lungs and the tissues. Recent studies indicated that O2 plays an important role in multiple erythrocyte properties, including membrane flexibility, glucose metabolism and volume control. Band 3 (also known as erythrocyte anion exchanger 1, AE1), the major human erythrocyte membrane-spanning protein, interacts with multiple proteins involved in the O2-dependent properties. Hemoglobin (Hb), the O2 carrier in erythrocytes, changes its conformation as a function of oxygen tension, leading to preferential interactions between band 3 and deoxyhemoglobin (deoxyHb), but not with oxyhemoglobin (oxyHb). The deoxyHb binding site of human band 3 has been mapped to residues 12-23, proximal to the binding sites for glycolytic enzymes and ankyrin, indicating that the binding of deoxyHb may interfere the interactions between band 3 and these proteins. Sequences on either side of the deoxyHb binding site have been shown to inhibit the binding of deoxyHb, indicating the band 3-deoxyHb association is carefully regulated. These observations raise the possibility that the O2-dependent reversible band 3-deoxyHb association may act as a molecular switch for the regulation of O2-sensitive properties in erythrocytes. To test this hypothesis in vivo, we generated three transgenic mouse models with altered band 3-deoxyHb interactions. In the first transgenic mouse model, the NH2-terminal 45 residues of mouse band 3 were displaced by the NH2-terminal 35 residues of human band 3 to generate a humanized deoxyHb binding site. The resulting mouse provides us with a quasi-human model for analysis of the regulatory role of deoxyHb binding to band 3 on RBC properties. In the second transgenic mouse, the same human sequence was inserted into the murine band 3, except the amino acids responsible for deoxyHb binding (residues 12-23) were deleted. This mouse model enables us to determine how the absence of a deoxyHb binding site on band 3 might affect O2 regulation of RBC properties. In the third transgenic mouse, the deoxyHb binding site has been mutated (deletion of residues 1-11) to exhibit supra-physiologically high affinity for deoxyHb, allowing us to determine how a sustained association of deoxyHb with band 3, even under fully oxygenated conditions, might impact O2 modulation of RBC biology. Investigation of the O2-dependent erythrocyte properties, including band 3-ankyrin association, glycolytic enzyme assembly, and ion transport in erythrocytes from these transgenic mice confirmed that the reversible band 3-deoxyHb interaction controls the regulation of the O2-dependent properties of erythrocytes. As expected, without the deoxyHb binding site on band 3, erythrocyte properties do not respond to changes of O2 tension. In the erythrocytes with the high affinity deoxyHb binding site on band 3, glycolytic enzymes and ankyrin are displaced even under high O 2 tension equals to that in the arteries. In addition, we discovered a novel mechanism of O2-dependent regulation of the Na+-K+-2Cl– cotransporter (NKCC) in erythrocytes. Although it was known that O2 can regulate the activity of NKCC, the mechanism was not clear since no interaction between band 3 and related proteins had been discovered. Here, I report a novel interaction between band 3 and WNK1 (With No Lysine kinase-1), a serine/threonine kinase known to activate OSR1 (oxidative stress responsive kinase-1) which in turn phosphorylates and activates NKCC activity by multiple stimuli. Deletion of either residues 1-11 or 12-23 in band 3 inhibits the binding of WNK1, increases the activity of NKCC and reduces the O2-dependency. These findings suggest that the reversible band 3-deoxyHb association may also serve as a molecular switch for the regulation of NKCC activity by affecting the band 3-WNK1 interaction. We further mapped the binding sites on both band 3 and WNK1 and concluded that WNK1 interacts with the NH2-terminal of band 3 through its COOH-terminal. In conclusion, our discoveries confirm the hypothesis that the O 2-dependent reversible association between band 3 and deoxyHb serves as a molecular switch to regulate the O2-dependent properties in red blood cells by affecting interactions between band 3 and other key proteins.