Southeast Asian AE1 associated renal tubular acidosis: Cation leak is a class effect
Introduction
The anion exchanger, AE1 (or Band 3), encoded by the gene SLC4A1, is a membrane bound glycoprotein that is predicted to span the cell membrane 12–14 times [1]. It mediates the electroneutral exchange of chloride for bicarbonate across the cell membrane, and is present chiefly in the erythrocyte, (where it is highly expressed, at approximately 1 × 106 copies per cell), and in the α-intercalated cell of the kidney.
Its function in the red cell is to reclaim bicarbonate from the red cell in the peripheral circulation after CO2 absorption by the erythrocyte. An isoform, truncated by 65 amino acids at the N-terminal end of the protein is present in the basolateral membrane of the acid-secreting α-intercalated cell [2], to reclaim bicarbonate back into the systemic circulation following the hydration of CO2 catalyzed by carbonic anhydrase in the intercalated cell interior.
Distinct mutations of the SLC4A1 gene may cause disease of either the red cell or the kidney. Certain mutations of SLC4A1 can cause red cell diseases such as hereditary spherocytosis (HS) [3], hereditary stomatocytosis (HSt) [4] and Southeast Asian ovalocytosis (SAO) [5]. In these last two diseases, the defect is often functional-the AE1 protein having reduced efficiency at anion exchange when expressed in Xenopus oocytes [4], [5].
Other mutations can cause kidney disease, namely distal renal tubular acidosis (dRTA), a failure of the α-intercalated cell resulting in faulty urinary acidification, resulting in varying degrees of systemic acidosis, renal stone formation, bone demineralisation and hypokalemia. These mutations usually result in proteins which have normal anion exchange activity when expressed in Xenopus oocytes, but are mistargetted in polarized mammalian cell models [6].
There are two phenotypes of AE1 associated dRTA; the rare European autosomal dominant (AD) dRTA, where one mutant allele will generate a mutant protein that is able to ‘capture’ a wild-type protein in the cytosol when heterodimerized with it, when expressed in a polarized cell model [7]. The other is comprised of the commoner Southeast Asian autosomal recessive (AR) dRTA, in which one wild-type allele is capable of ‘rescuing’ a mutant protein when heterodimerized with it, resulting in cell membrane expression of the heterodimer [7], [8].
More recent work by our group has shown that when expressed in Xenopus laevis oocytes, dRTA-associated AE1 mutants are ‘cation leaky’. This effect is demonstrable in the three European AD mutants that we characterized (R589H, G609R and S613F), but much greater in G701D, the only SE Asian AR mutant that we examined [9].
As this disparity between the European and Asian mutant seemed so large, we decided to characterize three other described SE Asian mutants (S773P, Δ850 and A858D) [10], [11], [12]., alongside the G701D mutant, using the X. laevis expression system. Our results show that these three other AE1 mutations also induce a cation leak in a still functional anion exchanger. The effect of these mutations on the anion exchange structure of the protein is then discussed as the consequences of this cation leak in cells expressing AE1.
Section snippets
Methods and materials
Plasmid and DNA construction. The pSP65erythroid human AE1 (hAE1) plasmid was used to construct the kidney AE1 (kAE1), deleting the 65 N-terminal amino acids, as described [9]. For point mutations, primers were used with the following codon substitutions:
S773P: TCC/CCC, ΔV850: GTG was deleted, A858D: GCC/GAC. Primers were purchased from Eurogentec (Seraing, Belgium); polymerase chain reactions (PCRs) were performed using a Biometra “UNO Thermoblock” thermocycler (Göttingen, Germany). Sequences
Western blot
Mutant kAE1 constructs were co-expressed in oocytes with the AE1 chaperonin glycophorin A (GPA), because without GPA there is absent membrane expression of G701D-kAE1 in oocytes [12]. Oocyte expression of wild-type and mutant kAE1 was confirmed by detecting bands of the appropriate molecular mass on western blotting (Fig. 1). The western blots were done at the same time as the functional studies, .the similar western blot pattern between the wild-type and mutant AE1 in the context of the
Discussion
We have demonstrated that in addition to G701D the three other AR dRTA AE1 associated mutants (S773P, ΔV85O and A858D) are all functional anion exchangers and all have a ‘cation leak’ that is of a similar magnitude. We chose these mutants on the basis that they have autosomal recessive phenotypes and were all found in Southeast Asia, just like the G701D mutation in which we demonstrated a large cation leak [9]. S773P was found in Northeast Thailand [11], the same region that G701D is prevalent
Acknowledgments
This work was supported by the European Dialysis and Transplant Association (EDTA-ERA) and the St. Peter’s Trust for Kidney, Bladder and Prostate Research (S.W.). Special thanks are offered to Professor Robert Unwin for his guidance and Professor John Cunningham for both his support and encouragement.
References (23)
- et al.
Novel topology in C-terminal region of the human plasma membrane anion exchanger, AE1
J. Biol. Chem.
(2003) - et al.
Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis
Blood
(1995) - et al.
Point mutations involved in red cell stomatocytosis convert the electroneutral anion exchanger 1 to a nonselective cation conductance
Blood
(2007) - et al.
Red blood cell band 3. Lysine 539 and lysine 851 react with the same H2DIDS (4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonic acid) molecule
J. Biol. Chem.
(1994) - et al.
The substrate anion selectivity filter in the human erythrocyte Cl−/HCO3-exchange protein, AE1
J. Biol. Chem.
(2004) - et al.
An inverting basket model for AE1 transport
J. Theor. Biol.
(2002) - et al.
Band 3 mutations, distal renal tubular acidosis, and Southeast Asian ovalocytosis
Kidney Int.
(2002) - et al.
South-east asian ovalocytic (SAO) erythrocytes have a cold sensitive cation leak: implications for in vitro studies on stored SAO red cells
Biochim. Biophys. Acta
(1999) - et al.
Invasion of hereditary ovalocytes by Plasmodium falciparum in vitro and its relation to intracellular ATP concentration
Mol. Biochem. Parasitol.
(1992) - et al.
Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+ATPase
Proc. Natl. Acad. Sci. USA
(1989)
Monovalent cation leaks in human red cells caused by single amino-acid substitutions in the transport domain of the band 3 chloride-bicarbonate exchanger, AE1
Nat. Genet.
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Structural model of the anion exchanger 1 (SLC4A1) and identification of transmembrane segments forming the transport site
2013, Journal of Biological ChemistryCitation Excerpt :A few years ago, some specific point mutations were characterized and proposed to convert the electroneutral anion exchanger into a cation-conductive pathway (5, 6). These mutations are associated with human pathologies (hereditary hemolytic anemia and distal renal tubular acidosis) (7–10). In addition to naturally occurring mutations identified in patients with hereditary stomatocytosis or distal renal tubular acidosis, a sequence-function analysis of AE1 revealed other amino acids crucial for transport properties of the protein (11).
Dual transport properties of anion exchanger 1: The same transmembrane segment is involved in anion exchange and in a cation leak
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2010, International Journal of Biochemistry and Cell BiologyCitation Excerpt :One of us has made a major study of the various mutant alleles that cause RTA in SE Asia. It turns out that all of them code for extremely cation-leaky proteins, in contrast to the dRTA causing mutations found in European populations (Walsh et al., 2008, 2009). This brings the list to 10: our HSt examples (L687P, D705Y, S731P, H734R, R760Q), SAO (deletion of 400–408) and these (S773P, G701D, Δ850, A858D).
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