The GPA-dependent, spherostomatocytosis mutant AE1 E758K induces GPA-independent, endogenous cation transport in amphibian oocytes

1 Divisions of Nephrology and Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts; and 2 Division of Perinatal Medicine, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut Submitted 6 October 2009 ; accepted in final form 5 November 2009 The previously undescribed heterozygous missense mutation E758K was discovered in the human AE1/SLC4A1/band 3 gene in two unrelated patients with well-compensated hereditary spherostomatocytic anemia (HSt). Oocyte surface expression of AE1 E758K, in contrast to that of wild-type AE1, required coexpressed glycophorin A (GPA). The mutant polypeptide exhibited, in parallel, strong GPA dependence of DIDS-sensitive 36 Cl – influx, trans-anion-dependent 36 Cl – efflux, and Cl – /HCO 3 – exchange activities at near wild-type levels. AE1 E758K expression was also associated with GPA-dependent increases of DIDS-sensitive pH-independent SO 4 2– uptake and oxalate uptake with altered pH dependence. In marked contrast, the bumetanide- and ouabain-insensitive 86 Rb + influx associated with AE1 E758K expression was largely GPA-independent in Xenopus oocytes and completely GPA-independent in Ambystoma oocytes. AE1 E758K-associated currents in Xenopus oocytes also exhibited little or no GPA dependence. 86 Rb + influx was higher but inward cation current was lower in oocytes expressing AE1 E758K than previously reported in oocytes expressing the AE1 HSt mutants S731P and H734R. The pharmacological inhibition profile of AE1 E758K-associated 36 Cl – influx differed from that of AE1 E758K-associated 86 Rb + influx, as well as from that of wild-type AE1-mediated Cl – transport. Thus AE1 E758K-expressing oocytes displayed GPA-dependent surface polypeptide expression and anion transport, accompanied by substantially GPA-independent, pharmacologically distinct Rb + flux and by small, GPA-independent currents. The data strongly suggest that most of the increased cation transport associated with the novel HSt mutant AE1 E758K reflects activation of endogenous oocyte cation permeability pathways, rather than cation translocation through the mutant polypeptide. chloride-bicarbonate exchange; Xenopus oocytes; Ambystoma oocytes; erythrocyte band 3, glycophorin A Address for reprint requests and other correspondence to the co-senior authors: S. L. Alper, Beth Israel Deaconess Medical Center, 330 Brookline Ave., E/RW763, Boston,