A Ser725Arg mutation in Band 3 abolishes transport function and leads to anemia and renal tubular acidosis

TO THE EDITOR:

Band 3, the anion exchanger (AE1) encoded by SLC4A1, is expressed in the erythrocyte membrane and in the collecting ducts of the kidney where it mediates transmembrane electroneutral chloride-bicarbonate exchange and serves as a linker between the plasma membrane and the cytoskeleton. Despite determination of the crystal structure of the human Band 3 membrane domain, specific amino acids that mediate anion binding and exchange remain largely unknown.¹  Studying human variants of AE1 may provide insights into the specific amino acids required for transport. In this study, we determined that a homozygous mutation, Ser725Arg, located in the anion-binding site of Band 3, causes anemia and distal renal tubular acidosis with loss of anion transport function likely due to an anomalous ionic interaction with the essential residue Glu681.

Heterozygous mutations in Band 3 have been linked to several phenotypes, including hereditary spherocytosis (HS), a subtype of hereditary cryohydrocytosis, Southeast Asian ovalocytosis (SAO), acanthocytosis, and distal renal tubular acidosis (dRTA). In a few cases, patients exhibit both hemolytic anemia and dRTA, typically with homozygous or compound heterozygous mutations associated with Band 3 deficiency due to abnormal trafficking from the endoplasmic reticulum (ER) to the plasma membrane.^2-6 

A 6-day-old male infant presented with tachypnea, intermittent hypoxia, scleral icterus, splenomegaly, and severe anemia, hemoglobin 6.6 g/dL, and marked spherocytosis. Serum bicarbonate was 17 mEq/L with anion gap 10 (Figure 1A). Erythrocytes demonstrated increased osmotic fragility and decreased eosin 5-maleimide (EMA) binding, a marker of membrane Band 3 content (Figure 1B-C). Monthly transfusions were required during the first year of life. Growth was poor, at the third percentile.

The parents are first cousins from Pakistan. There was no family history of hematologic disease. Hematologic indices, mildly increased osmotic fragility, and mildly decreased EMA binding indicate that both parents have compensated HS (Figure 1).

At 6 months of age, the proband’s serum bicarbonate was 16 mEq/L, urine pH was 8.0, and renal ultrasound revealed small kidneys with diffuse, bilateral nephrocalcinosis, findings suggestive of dRTA. Urine electrolytes confirmed positive anion gap. Sodium citrate was prescribed with good benefit. Serum bicarbonate increased, and weight gain improved. In the first, second, and third years of life, the patient received 13, 6, and 6 transfusions, respectively. In the fourth year of life, the patient maintained hemoglobin at ∼7.5 g/dL, with transfusions given twice for worsening anemia during intercurrent illness.

Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) of erythrocyte membrane proteins from the proband (pretransfusion), his mother, and his father revealed Band 3 deficiency (32%, 61%, and 48% of control, respectively). Next-generation sequencing identified a homozygous A-to-C substitution in the SLC4A1 gene (nucleotide position chr17:42330624), changing Ser (AGT) to Arg (CGT) at residue 725 (NM_000342:c.A2173C:p.S725R; Figure 1A). This SLC4A1 missense mutation was predicted to be highly pathogenic (CADD Phred score 28.4) and damaging by SIFT, PolyPhen2, and other mutation prediction algorithms. It is novel and unique, not present in the National Heart, Lung, and Blood Institute Exome Sequencing Project database, the 1000 Genomes database, or the ExAC (>60 000 exomes) database.

Both parents were heterozygous for the S725R variant, which we name Band 3^Punjab. These studies were approved by the Yale University School of Medicine institutional review board (12377).

Ser725 is a highly conserved residue in SLC4A1 proteins across vertebrates, clades of placental mammals, the extant Eutherians, birds, reptiles, and frogs, with a highly significant vertebrate phastCons score of 1.0 and a highly significant PhyloP conservation score of 1.06 (Figure 1D). However, it is not conserved among other members of the SLC4 transporters, being Ala in SLC4A2, Thr in SLC4A3, a hydrophobic residue in the sodium bicarbonate cotransporters, and His in SLC4A11 (Figure 1E).

The crystal structure of the membrane domain of Band 3 shows that Ser725 is located in an extended region of transmembrane (TM) 10 just NH₂-terminal to a half-helix beginning at Thr728 (Figure 1F).¹  The NH₂-termini of TM10 and TM3 helices face each other in the middle of the protein, and their positive helix dipoles are likely involved in binding anions.⁷  TM10 also contains Arg730, an essential residue that points back toward the anion-binding site. The side chain of Ser725 is only 3 Å away from Glu681 in TM8, the proton-binding site required for sulfate uptake.^8,9  Mutation of the homologous glutamate in murine AE1, E669Q, abrogated chloride-mediated anion exchange.^10,11  This suggests that Ser725 is located within the anion-binding site of Band 3 and that there could be a hydrogen-bonding interaction between these 2 residues. Molecular modeling predicts that S725R mutation would result in the formation of an ionic bond between Arg725 and Glu681, thereby impairing anion transport.

Transport function was assessed in a Cl⁻/HCO₃⁻ exchange assay in HEK293 cells transfected with Band 3 or an external HA-tagged membrane domain (md) of Band 3 with wild-type (WT), S725R, S725A, or pcDNA (Thermo-Fisher) empty vector control, as has been described.^12,13  Fluorescence measurements were converted to intracellular pH (pH_i) by the nigericin-high potassium method, and anion-exchange activity was calculated after protein expression level normalization.¹⁴  Normalized transport activity of Band 3 variants was calculated as H⁺-equivalent flux/Band 3 cellular abundance = H⁺-equivalent flux/(Band 3 intensity/transcription factor II D [TFIID] intensity). The S725A mutant in the Band 3 construct had normal expression levels, as did the S725R mutant, whereas the S725R mutant in the mdAE1 construct was expressed at ∼50% the level of WT. Anion transport was completely abolished in the S725R mutant in both Band 3 and the membrane domain construct, whereas the S725A mutants had normal transport activity (Figure 2A-B).

Immunofluorescence microscopy using an anti–human influenza hemagglutinin antibody revealed HEK cells transfected with either the S725R or S725A mutant demonstrated staining at the cell surface in intact cells,^15,16  confirming trafficking of the mutants to the plasma membrane (Figure 2C). Permeabilized cells revealed intracellular protein localized to the ER; the overlap in staining at the cell surface was used to estimate the cell surface expression levels. The S725A mutant and the S725R mutant were expressed at the cell surface at 90% and 60% of the percentage found for the wild-type protein, consistent with decreased endogenous expression observed in EMA binding and SDS-PAGE in mutant erythrocytes. Thus, although the S725R mutant can traffic to the cell surface, it lacks any transport activity.

Homozygous mutations in Band 3 have led to different effects on membrane levels of Band 3 and anion exchange in the erythrocyte and kidney but have not shed light on specific transport-related residues.^2-6  Band 3^Coimbra, V488M, and Band 3^Vienna, S477X, lead to complete loss of membrane-associated Band 3, resulting in transfusion dependence and dRTA.^2,6  Patients with homozygous A858D exhibit acanthocytic anemia and dRTA,^4,17,18  whereas homozygous Band 3^Courconnes leads to HS and incomplete dRTA,³  with both mutants exhibiting ER retention leading to decreased membrane Band 3. Homozygous Band 3^Neapolis, a splicing mutation 5′ to the kidney Band 3 isoform, leads to erythrocyte membrane Band 3 deficiency and severe HS but no dRTA.¹⁹  Homozygous Band 3^SAO, in-frame deletion of 9 amino acids at the cytosolic and membrane domain hinge region causing severe misfolding, leads to transfusion-dependent anemia and RTA.⁵ 

The S725R mutant leads to Band 3 protein deficiency and exhibits near-complete lack of ion transport function, likely due to the formation of a salt bridge between the positively charged arginine at 725 and the nearby Glu681. Decreased Band 3 expression and lack of transport activity lead to anemia and dRTA, respectively, in the homozygous state.

These results raise the question of whether anion exchange influences erythrocyte membrane integrity and function. In vitro studies of human erythrocytes demonstrate that under conditions of complete inhibition of anion transport by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), ∼10% of spectrin-ankyrin membrane complexes are not extractable, suggesting a possible role for anion transport in Band 3–cytoskeletal interactions.²⁰  Cows and mice with total lack of Band 3 are viable, suffering from spherocytosis and dRTA.^21-23  Band 3–deficient cows exhibit mild acidosis, worsened after exercise or dietary acid load, with bicarbonate concentration and total CO₂ content in the low-normal range. CO₂ saturation of blood is infrequent; thus the additional CO₂ load facilitated by Band 3 is likely not critical except under very high stress conditions. Demonstration of elevated total CO₂ in Band 3–deficient erythrocytes lacking anion transport would begin to address these questions.