Regions of human kidney anion exchanger 1 (kAE1) required for basolateral targeting of kAE1 in polarised kidney cells: mis-targeting explains dominant renal tubular acidosis (dRTA)

Abstract

Distal renal tubular acidosis (dRTA) is characterised by defective acid secretion by kidney α-intercalated cells. Some dominantly inherited forms of dRTA result from anion exchanger 1 (AE1) mutations. We have developed a stably transfected cell model for the expression of human kidney AE1 (kAE1) and mutant kAE1 proteins in MDCKI cells. Normal kAE1 was delivered to the plasma membrane of non-polarised cells and to the basolateral membrane of polarised cells. The AE1 N-glycan was processed to a complex form. Surprisingly, expression of kAE1 increased the permeability of the paracellular barrier of polarised MDCKI monolayers. All dominant dRTA mutations examined altered the targeting of kAE1 in MDCKI cells. The mutant proteins kAE1(R589H), kAE1(S613F) and kAE1(R901Stop) were retained in the ER in non-polarised cells, but the kAE1(R901Stop) protein was also present in late endosomes/lysosomes. The complex N-glycan of kAE1(R901Stop) was larger than that of normal kAE1. In polarised cells, the mutant kAE1(R901Stop) was mis-targeted to the apical membrane, while the kAE1(R589H) and kAE1(S613F) mutants did not reach the cell surface. These results demonstrate that dominant dRTA mutations cause aberrant targeting of kAE1 in polarised kidney cells and provide an explanation for the origin of dominant dRTA. Our data also demonstrate that the 11 C-terminal residues of kAE1 contain a tyrosine-dependent basolateral targeting signal that is not recognised by μ1B-containing AP-1 adaptor complexes. In the absence of the N-terminus of kAE1, the C-terminus was not sufficient to localise kAE1 to the basolateral membrane. These results suggest that a determinant within the kAE1 N-terminus co-operates with the C-terminus for kAE1 basolateral localisation.

Regulated acid–base transport in the collecting duct

“…The S613F mutant is predicted from cell culture models to lead to partially misrouted apical AE1 expression (188). In contrast, no luminal expression of AE1 was detected in the kidney from a patient carrying the S613F mutation (210).…”

“…Mechanistically, these mutations may affect the polarized localization of AE1 at the basolateral membrane of type A intercalated cells, remain intracellular, or lose activity (6,7,39,47,93,141,150,188,189). The autosomal dominant pattern of inheritance in certain mutants is possibly due to the fact that the transporter dimerizes or that partial rerouting of mutant AE1 to the luminal membrane of type A intercalated cells may shunt normal acid secretion.…”

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.…”

Analysis of the interaction between human kidney anion exchanger 1 and kanadaptin using yeast two-hybrid systems

“…Unlike wild-type kAE1, the mutant proteins exhibited intracellular retention when they were studied in non-polarized cells (Quilty et al, 2002a;Quilty et al, 2002b;Toye et al, 2002;Kittanakom et al, 2004a). However, in polarized cells, dRTA mutations cause either intracellular retention (Toye et al, 2004) or apical mistargeting of the proteins (Devonald et al, 2003;Toye et al, 2004;Rungroj et al, 2004). The correct trafficking of kAE1 may require both N-and C-termini (Devonald et al, 2003;Toye et al, 2004).…”

“…However, in polarized cells, dRTA mutations cause either intracellular retention (Toye et al, 2004) or apical mistargeting of the proteins (Devonald et al, 2003;Toye et al, 2004;Rungroj et al, 2004). The correct trafficking of kAE1 may require both N-and C-termini (Devonald et al, 2003;Toye et al, 2004). Tyrosine-based sorting signals locating at these regions may interact with adaptor-protein complexes (Bonifacino and Dell'Angelica, 1999;Mostov et al, 2000).…”