4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Spherocytosis--Hereditary

We identified 11 human pedigrees with dominantly inherited hemolytic anemias in both the hereditary stomatocytosis and spherocytosis classes. Affected individuals in these families had an increase in membrane permeability to Na and K that is particularly marked at 0 degrees C. We found that disease in these pedigrees was associated with a series of single amino-acid substitutions in the intramembrane domain of the erythrocyte band 3 anion exchanger, AE1. Anion movements were reduced in the abnormal red cells. The 'leak' cation fluxes were inhibited by SITS, dipyridamole and NS1652, chemically diverse inhibitors of band 3. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na and K fluxes in the oocytes, and the induced Cl transport was low. These data are consistent with the suggestion that the substitutions convert the protein from an anion exchanger into an unregulated cation channel.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amino Acid Substitution; Anemia, Hemolytic; Animals; Anion Exchange Protein 1, Erythrocyte; Benzoates; Biological Transport; Cations; Cell Membrane Permeability; Chlorides; Dipyridamole; Erythrocytes; Humans; Molecular Sequence Data; Oocytes; Pedigree; Phenylurea Compounds; Phosphodiesterase Inhibitors; Potassium; Protein Structure, Tertiary; RNA; Sodium; Spherocytosis, Hereditary; Xenopus laevis

Hereditary spherocytosis (HS) is a common inherited hemolytic anemia caused by mutations in erythrocyte proteins including the anion exchanger, AE1 (band 3). This study examined seven missense mutations (L707P, R760Q, R760W, R808C, H834P, T837M, and R870W) located in the membrane domain of the human AE1 that are associated with this disease. The HS mutants, constructed in full-length AE1 cDNA, could be transiently expressed to similar levels in HEK 293 cells. Immunofluorescence, cell surface biotinylation, and pulse chase labeling showed that the HS mutants all exhibited defective cellular trafficking from the endoplasmic reticulum to the plasma membrane. Impaired binding to an inhibitor affinity matrix indicated that the mutant proteins had non-native structures and may be misfolded. Further characterization of the HS R760Q mutant showed no change in its oligomeric structure or turnover (half-life = 15 h) compared to wild-type AE1, suggesting the mutant was not aggregated or targeted for rapid degradation via the proteasome. Intracellular retention of HS mutant AE1 would lead to destruction of the protein during erythroid development and would account for the lack of HS mutant AE1 in the plasma membrane of the mature red cell.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Biotinylation; Cell Line; Chromatography, Affinity; Chromatography, High Pressure Liquid; Dimerization; Erythrocytes; Glycosylation; Golgi Apparatus; Humans; Immunoblotting; Mutation, Missense; Protein Folding; Protein Structure, Tertiary; Protein Transport; Spherocytosis, Hereditary; Transfection