Mutation Conferring Apical-Targeting Motif on AE1 Exchanger Causes Autosomal Dominant Distal RTA

Abstract

Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the a-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1.

Structure, Function, and Trafficking of SLC4 and SLC26 Anion Transporters

“…Insertion of single base results in frameshift and novel 15-amino acid C-terminal sequence to residue 919 (Shao et al, 2010) M909T Dominant Functional Mislocalized to both apical and basolateral membranes (Fry et al, 2012) as five amino acids from the C-terminus of AE1 impaired ER exit, while deletion of 20 residues profoundly decreased protein stability and expression (Cordat, Li, & Reithmeier, 2003). In agreement with these findings, some of the latest dRTA mutations identified are either point or frameshift mutations within the short carboxyl-terminus of AE1 (Fry et al, 2012;Shao et al, 2010;Zhang et al, 2012).…”

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

“…41 Furthermore, we recently identified a novel dRTA-causing mutation, kAE1-M909T, which lies within the potential class II PDZ-binding domain (A 908 MPV) and converts it to class I (A 908 TPV). 16 The M909T mutant protein showed a nonpolarized pattern, and deletion of the A 908 MPV motif resulted in intracellular retention of the kAE1-D4 mutant protein in MDCK cells, indicating that this motif is critical for normal basolateral localization of kAE1. 16 However, the actual PDZ proteins involved are not known.…”

“…16 The M909T mutant protein showed a nonpolarized pattern, and deletion of the A 908 MPV motif resulted in intracellular retention of the kAE1-D4 mutant protein in MDCK cells, indicating that this motif is critical for normal basolateral localization of kAE1. 16 However, the actual PDZ proteins involved are not known. On the basis of these data, it is very likely that there are multiple sorting motifs that determine kAE1's membrane trafficking, retention, and recycling.…”

“…In addition, it is notable that AE1 mutations associated with autosomal dominant dRTA are spread over both transmembrane and C-terminal domains, but no such mutations have thus far been identified within the N terminus. The C-terminal tail of AE1 is composed of 36 residues; this domain includes three reported dRTA-causing mutations: A888L+889X, 15 M909T, 16 and R9013. 17 R901X and M909T mutant proteins expressed in Xenopus oocytes exhibit preserved anion transport function, but both are mistargeted to the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.…”

“…17 R901X and M909T mutant proteins expressed in Xenopus oocytes exhibit preserved anion transport function, but both are mistargeted to the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells. 5,6,16 As a consequence, we and others have sought identification of novel binding partners for this domain, to ascertain molecular basis of sorting, trafficking, and membrane retention of kAE1. Several proteins are reported to interact with the C terminus of kAE1, and disruption of these interactions results in abnormal cellular location of kAE1.…”