Band 3 Missense Mutations and Stomatocytosis: Insight into the Molecular Mechanism Responsible for Monovalent Cation Leak

Abstract

Missense mutations in the erythroid band 3 protein (Anion Exchanger 1) have been associated with hereditary stomatocytosis. Features of cation leaky red cells combined with functional expression of the mutated protein led to the conclusion that the AE1 point mutations were responsible for Na+ and K+ leak through a conductive mechanism. A molecular mechanism explaining mutated AE1-linked stomatocytosis involves changes in AE1 transport properties that become leaky to Na+ and K+. However, another explanation suggests that point-mutated AE1 could regulate a cation leak through other transporters. This short paper intends to discuss these two alternatives.

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.…”

“…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. The defect in Cl Ϫ /HCO 3 Ϫ transport activity of G796R and R730C mutants was not due to reduced expression or processing of the proteins to the cell surface and also corroborates their inactivity reported in Xenopus oocytes (4,15,28). The inducible system used is a valuable tool to control AE1 expression in HEK293 cells, and the heterogeneous amounts of the protein at cell surface from one sample to the other actually validated our method based on the comparison of activities.…”

“…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.…”

“…Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na ϩ and K ϩ fluxes. Intracellular pH measurements using microelectrodes, 36 Cl Ϫ influx, and 35 SO 4 Ϫ2 efflux in oocytes showed that HSt mutant AE1 proteins were no more functional, except for E758K and R760Q mutants that kept anion exchange activity, although cation leaks were also detected in these two variants.…”

“…11) or hereditary distal renal tubular acidosis (1). Recently, nine point mutations in the SLC4A1 gene (L687P, D705Y, R730C, S731P, H734R, E758K, R760Q, S762R, and G796R substitutions in the protein) have been reported (4,7,13,14,15,28,30). All these substitutions induce monovalent cation leaks in RBCs and are responsible for hereditary stomatocytosis (HSt) associated with hemolytic anemia (6).…”