Dominant and Recessive Distal Renal Tubular Acidosis Mutations of Kidney Anion Exchanger 1 Induce Distinct Trafficking Defects in MDCK Cells

Abstract

Distal renal tubular acidosis (dRTA), a kidney disease resulting in defective urinary acidification, can be caused by dominant or recessive mutations in the kidney Cl -/HCO 3 -anion exchanger (kAE1), a glycoprotein expressed in the basolateral membrane of a-intercalated cells. We compared the effect of two dominant (R589H and S613F) and two recessive (S773P and G701D) dRTA point mutations on kAE1 trafficking in Madin-Darby canine kidney (MDCK) epithelial cells. In contrast to wild-type (WT) kAE1 that was localized to the basolateral membrane, the dominant mutants (kAE1 R589H and S613F) were retained in the endoplasmic reticulum (ER) in MDCK cells, with a few cells showing in addition some apical localization. The recessive mutant kAE1 S773P, while misfolded and largely retained in the ER in non-polarized MDCK cells, was targeted to the basolateral membrane after polarization. The other recessive mutants, kAE1 G701D and designed G701E, G701R but not G701A or G701L mutants, were localized to the Golgi in both non-polarized and polarized cells. The results suggest that introduction of a polar mutation into a transmembrane segment resulted in Golgi retention of the recessive G701D mutant. When coexpressed, the dominant mutants retained kAE1 WT intracellularly, while the recessive mutants did not. Coexpression of recessive G701D and S773P mutants in polarized cells showed that these proteins could interact, yet no G701D mutant was detected at the basolateral membrane. Therefore, compound heterozygous patients expressing both recessive mutants (G701D/S773P) likely developed dRTA due to the lack of a functional kAE1 at the basolateral surface of a-intercalated cells.

Distal Renal Tubular Acidosis in Mice Lacking the AE1 (Band3) Cl−/HCO3 − Exchanger (slc4a1)

“…kAE1 forms functional homodimers or hetero-oligomers, and oligomerization of a dominant negative mutant AE1 with a wild-type polypeptide in the heterozygote A-IC likely explains some dominant disease. The mutant/ wild-type heterodimer either fails to traffic to the cell surface or, in some cases, traffics normally but may not function normally at the cell surface (18,61,62). Another subset of kAE1 mutant polypeptides seems to cause dominant dRTA by inappropriate targeting to A-IC apical membrane, with resultant apical bicarbonate secretion likely short-circuiting luminal acid secretion (19,20,63).…”

“…At least two pathophysiologic mechanisms have been proposed to explain autosomal dominant dRTA as a result of AE1 mutations (1,9): A dominant negative intracellular retention phenotype (18) and a dominant or co-dominant mistargeting to the apical membrane (19,20). A distinct set of mutations found among Southeast Asians and Melanesians is transmitted in autosomal or compound recessive patterns (11,14).…”

Physical and Functional Links between Anion Exchanger-1 and Sodium Pump

“…Identification of the b1/AE1 interaction indicates a direct link, and a potential regulatory interaction, between important anion and cation transporters in the kidney. likely to be the result of chronic metabolic acidosis, because a similar b1 reduction is not apparent after oral NH 4 Cl loading in WT animals (red in g and h). Bar, 10 mm.…”

“…To date, at least two mistargeting phenotypes, intracellular retention and aberrant membrane accumulation, have been observed and are the major pathogenic mechanisms. [4][5][6] AE1 is composed of a large cytosolic N-terminal domain, a central transmembrane region that is predicted to span the lipid bilayer 12-14 times and is responsible for the anion exchange, and a short cytosolic C-terminal tail (AE1C). Multiple binding sites for other proteins including cytoskeletal proteins ankyrin-1 (ANK1), glycolytic enzymes, and hemoglobin have been identified in the N-terminal domain of eAE1.…”

Evidence of a Structural and Functional Ammonium Transporter RhBG·Anion Exchanger 1·Ankyrin-G Complex in Kidney Epithelial Cells

“…HEK293 cells, lacking endogenous expression of kAE1 (22), were stably transfected with wild-type or mutant kAE1 cDNA using the tetracycline-inducible expression system previously described (19) to circumvent the loss of long term expression of AE1 protein (23,24). Flow cytometry experiments using BRIC6 antibody revealed wild-type kAE1 at the surface of tetracyclineinduced transfected cells ( Fig.…”