Mild spherocytosis and altered red cell ion transport in protein 4.2–null mice
Luanne L. Peters, … , David E. Golan, Carlo Brugnara
Luanne L. Peters, … , David E. Golan, Carlo Brugnara
Published June 1, 1999
Citation Information: J Clin Invest. 1999;103(11):1527-1537. https://doi.org/10.1172/JCI5766.
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Article

Mild spherocytosis and altered red cell ion transport in protein 4.2–null mice

Abstract

Protein 4.2 is a major component of the red blood cell (RBC) membrane skeleton. We used targeted mutagenesis in embryonic stem (ES) cells to elucidate protein 4.2 functions in vivo. Protein 4.2–null (4.2–/–) mice have mild hereditary spherocytosis (HS). Scanning electron microscopy and ektacytometry confirm loss of membrane surface in 4.2–/– RBCs. The membrane skeleton architecture is intact, and the spectrin and ankyrin content of 4.2–/– RBCs are normal. Band 3 and band 3–mediated anion transport are decreased. Protein 4.2–/– RBCs show altered cation content (increased K+/decreased Na+)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 cell shrinkage. We conclude that protein 4.2 is important in the maintenance of normal surface area in RBCs and for normal RBC cation transport.

Authors

Luanne L. Peters, Hitesh K. Jindel, Babette Gwynn, Cathy Korsgren, Kathryn M. John, Samuel E. Lux, Narla Mohandas, Carl M. Cohen, Michael R. Cho, David E. Golan, Carlo Brugnara

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Figure 1

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Epb4.2 gene targeting results in a null mutation. (a) Restriction map of...
Epb4.2 gene targeting results in a null mutation. (a) Restriction map of the Epb4.2 locus with the targeted allele shown below. Numbered boxes represent exons. Upon homologous recombination in ES cells, a neomycin-resistant cassette (hatched box) replaced a segment of the gene extending from intron 3 to exon 8. A 667-bp 5′-flanking KpnI-NsiI fragment (filled bar) was used as the hybridization probe to distinguish alleles by Southern blot analysis. Kp, KpnI. Ns, NsiI. Xb, XbaI. Ec, EcoRI. Inset: Southern blot analysis. Homologous recombination occurred in 6 of 114 clones, as indicated by the presence of both the wild-type (18.5 kb) and targeted (14.9 kb) alleles in KpnI-digested DNA. (b) SDS-PAGE analysis (Fairbanks’ method) of RBC membranes (5 μg protein per lane). Protein 4.2 is undetectable and band 3 decreased in 4.2–/– RBCs. (c) Immunoblot analysis of RBC membrane ghosts (left; 5 μg protein per lane) and whole-cell lysates (right; 2.5 μL packed cells per lane). The blots were probed with a polyclonal antibody raised against purified human erythrocyte 4.2 (19). (d) Northern blot analysis of reticulocyte RNA from newborn mice (5 μg per lane). The filters were probed with a 2.4-kb mouse protein 4.2 cDNA (1). (e) Immunoblot of whole-platelet lysates probed with 4.2 antisera, as in c. Lane 1, 1.0 μg normal RBC membranes; lane 2, 3.0 μg normal platelet lysate; lane 3, 6.0 μg 4.2–/– platelet lysate. (f) RNase protection assay of kidney mRNA. Perfused kidney mRNA (10 μg per lane) was hybridized with a 295-bp 32P-labeled riboprobe corresponding to nucleotides 726–1019 (1) within the targeted region of protein 4.2 cDNA. In the left, middle, and right panels, RNase dilutions of 1:200, 1:100, and 1:50 were used, respectively. C is the RNA-free control lane. Sp, spectrin. Ank, ankyrin. bd 3, band 3. 4.1, protein 4.1. 4.2, protein 4.2. RBC, red blood cell ghost membranes. PLT, whole-platelet lysates.