IntroductionProtein 4.2 is a 72-kDa component of the red blood cell (RBC) membrane skeleton (200,000 copies per RBC) (1). An intact membrane skeleton attached to the lipid bilayer by vertical interactions between spectrin, ankyrin, protein 4.2, and band 3 (anion exchanger 1 [AE1]) is required for normal RBC membrane integrity. Defects in all of these components have been identified in hereditary spherocytosis (HS) (2). In humans, protein 4.2 defects are most common in people of Japanese descent and give rise to pleiotropic RBC morphological changes and hemolytic anemias of varying severity (3).In RBCs, protein 4.2 binds to the cytoplasmic domain of band 3 (4). In solution, protein 4.2 binds directly to spectrin, ankyrin, and protein 4.1, and promotes binding of spectrin to ankyrin-depleted inside-out vesicles (4, 5). The significance of these interactions in vivo is unknown. However, mice totally deficient in band 3 are also completely protein 4.2 deficient, suggesting that band 3 is critical for stable incorporation of protein 4.2 into the membrane skeleton (6).Hereditary spherocytes are characterized by increased Na + and decreased K + content compared with normal RBCs (2). Studies in mice and humans suggest that the cation alterations are the result of increased passive RBC membrane permeability and that the increased passive flux reflects the overall loss of membrane skeleton integrity, not the specific protein defect (7,8). Protein 4.2 has been proposed to negatively regulate band 3-mediated RBC anion transport (9), but its role in regulating RBC cation transport and intracellular cation content is unknown.We targeted the erythrocyte protein 4.2 gene (Epb4.2) in embryonic stem (ES) cells to create a null mutation (4.2 -/-) in mice. Deficiency of protein 4.2 in mice results in mild HS. Normal amounts of spectrin and ankyrin are assembled onto the membrane, and the membrane skeleton architecture is intact. The band 3 content of the membrane is decreased. Contrary to previous reports, band 3-mediated anion transport in not increased in 4.2 -/-RBCs, but is decreased. Protein 4.2-deficient RBCs show significant changes in RBC cation transporter activities. These changes are in marked contrast to those obtained in spectrin-and ankyrin-deficient mouse and human red cells (7,8), suggesting that specific membrane skeleton defects differentially influence cation transporter activities. Our results show that protein 4.2 is important in the maintenance of normal surface area in RBCs and is required for normal RBC cation transport. resulting in dehydration. The passive Na + permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4.2 -/-RBCs are significantly increased. Protein 4.2 -/-RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2 -/-RBCs compared with controls. The increased passive Na + permeability of 4.2 -/-RBCs is also dependent on c...