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

Abstract

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.

Rapid Cl−/HCO3− exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells

“…We could observe by stopped-flow spectrofluorometry a rapid intracellular alkalinization corresponding specifically to this exchange in HEK293 expressing WT AE1, whereas no activity was detectable in parental cells. Our results also showed that E758K mutant was fully active, as previously reported in Xenopus oocytes (4,29). Indeed, although the alkalinization rate constant was five to six times lower for E758K mutant than for WT AE1 (0.07 vs. 0.38 s Ϫ1 ), normalization of transport values with surface expression level indicated comparable relative activities (98 vs. 100%) and apparent unit permeabilities (11.96 ϫ 10 Ϫ3 vs. 9.81 ϫ 10 Ϫ3 m 3 /s) of both E758K mutant and normal proteins.…”

“…In the latter situation, affected individuals have an increase in RBC membrane permeability to cations and in most cases a reduction of anion movements through AE1. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na ϩ and K ϩ fluxes (4,7,13,14,15,28,29). These data were consistent with the concept that the substitutions convert the protein from an anion exchanger into an unregulated cation channel, although, so far, no study has clearly demonstrated that the cation leaks are mediated through AE1.…”

Missense mutations in the ABCB6 transporter cause dominant familialpseudohyperkalemia

“…These changes could lead either to cation leak through the normal substrate translocation pathway of ABCB6, or to the generation of a novel constitutive or cyclic leak pathway through the protein. Alternatively, mutant ABCB6 polypeptides could activate an independent cation permeability through direct or indirect protein-protein interaction, as proposed for CHC mutations in AE1/SLC4A1 [11,15], stomatin-deficient OHSt mutations in RhAG [12,13], and suggested by the fourfold elevation of K-Cl cotransport observed in red cells of patient FP Lille (LdeF, unpublished data).…”

“…These include SLC4A1 (an ion exchange) in CHC and in atypical forms of hereditary spherostomatocytosis [10,11], RHAG (Rh-associated glycoprotein) in isolated stomatin-deficient OHSt [12,13] GLUT1 (glucose transporter 1) in echinocytosis with paroxysmal dyskinesia [14], stomatin-deficient cryohydrocytosis [15] or in CHC [16] or pseudohyperkalemia and hemolysis [17] with neurological symptoms, and PIEZO1 (mechanosensitive cation channel protein FAM38A) in DHSt [18]. These findings suggest that distinct missense mutations in various red cell membrane solute transporters or channels generate cation leak pathways either through the mutant proteins themselves or by deregulating one or more independent cation permeabilities of the red cell membrane.…”

Novel Gardos channel mutations linked to dehydrated hereditary stomatocytosis (xerocytosis)

“…Mutations in several genes have been shown to cause red cell cation leak disorders. These include chloride-bicarbonate exchanger SLC4A1/AE1/ Band 3 in CHC and in atypical forms of hereditary spherostomatocytosis [23,24], ammonia transporter RHAG (Rh-associated glycoprotein) in isolated stomatin-deficient OHSt [25,26], glucose transporter GLUT1 in echinocytosis with paroxysmal dyskinesia [27], stomatin-deficient CHC [28] or in CHC [29] or pseudohyperkalemia and hemolysis [30] with neurological symptoms, and PIEZO1 (mechanosensitive cation channel protein FAM38A) in DHSt [10][11][12][13]. A recent study has highlighted a novel DHSt gene, KCNN4, encoding the widely expressed KCa3.1 Gardos channel, a Ca 21 -sensitive K 1 channel of intermediate conductance [14].…”