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

Abstract

Anion exchanger-1 (AE1) mediates chloride-bicarbonate exchange across the plasma membranes of erythrocytes and, via a slightly shorter transcript, kidney epithelial cells. On an omnivorous human diet, kidney AE1 is mainly active basolaterally in a-intercalated cells of the collecting duct, where it is functionally coupled with apical proton pumps to maintain normal acid-base homeostasis. The C-terminal tail of AE1 has an important role in its polarized membrane residency. We have identified the b1 subunit of Na + , K + -ATPase (sodium pump) as a binding partner for AE1 in the human kidney. Kidney AE1 and b1 colocalized in renal a-intercalated cells and coimmunoprecipitated (together with the catalytic a1 subunit of the sodium pump) from human kidney membrane fractions. ELISA and fluorescence titration assays confirmed that AE1 and b1 interact directly, with a K d value of 0.81 mM. GST-AE1 pull-down assays using human kidney membrane proteins showed that the last 11 residues of AE1 are important for b1 binding. siRNAinduced knockdown of b1 in cell culture resulted in a significant reduction in kidney AE1 levels at the cell membrane, whereas overexpression of kidney AE1 increased cell surface sodium pump levels. Notably, membrane staining of b1 was reduced throughout collecting ducts of AE1-null mouse kidney, where increased fractional excretion of sodium has been reported. These data suggest a requirement of b1 for proper kidney AE1 membrane residency, and that activities of AE1 and the sodium pump are coregulated in kidney.

PDLIM5 links kidney anion exchanger 1 (kAE1) to ILK and is required for membrane targeting of kAE1

“…5a) is one in which PDLIM5 physically tethers kAE1 to ILK, which would be required for correct movement of kAE1 towards its final basolateral membrane destination. Our previous studies of wild-type kAE1 in kidney epithelial cells also demonstrated that it is stabilized on the membrane by Na + , K + -ATPase through interaction of kAE1 with the pump’s β1 subunit21. Our current GST pulldown data (Fig.…”

“…In Xenopus oocytes, kAE1-Δ11 exhibits normal anion transport function, but is mis-targeted in both Madin-Darby Canine Kidney (MDCK) and HEK293 cells5618, supporting an initial idea that defective urinary acidification arises from trafficking defects of mutant proteins in kidney α-ICs1920. The C-terminal region is actually rich in potential targeting motifs/determinants21 including a PDZ ( P ost-synaptic density protein, PSD-95; D rosophila disc large tumour suppressor, Dlg; Z onula occludens-1, ZO-1) binding motif formed by the last 4 residues (A 908 MPV 911 ) that has not been previously characterized. The M909T mutation or deletion of the motif results in trafficking defects of the mutant proteins in polarized kidney epithelial cells, implicating this motif in proper membrane residency of kAE18.…”

“…Several proteins have so far been reported to associate with AE1C, and disruption of these interactions results in abnormal cellular location of kAE1212223. We report here a novel C-terminal binding partner, PDLIM5, also called Enigma homolog protein (ENH) and belonging to the Enigma subfamily of PDZ-LIM proteins.…”

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

“…Previous reports demonstrated a similar link between ankyrin binding and activity of an ion channel: ankyrin-G for cardiac Nav1.5 and neuronal Nav1.6 sodium channels (36,37) and ankyrin-B for cardiac K ATP channel (38). A very recent study showed a physical and functional interaction between kAE1 and Na ϩ ,K ϩ -ATPase in renal ␣-intercalated cells (39), and Na ϩ ,K ϩ -ATPase is known to bind ankyrin-G (26). The renal RhBG⅐kAE1⅐ankyrin-G complex described here, possibly including Na ϩ ,K ϩ -ATPase, is a new evidence of the great diversity of proteins that are organized in membrane microdomains in many cell types, under the control of ankyrins: ankyrin-G for neurofascin, KCNQ2/3, and voltage-gated Na ϩ channel in epithelial cells (40 -44) or E-cadherin and Na ϩ ,K ϩ -ATPase in axon initial segment (33,45) and ankyrin-B for Na ϩ ,K ϩ -ATPase, Na ϩ -Ca 2ϩ exchanger, and inositol triphosphate receptor in cardiomyocytes (46 -48) and ankyrin-R for Rh and eAE1 in RBCs (4,6).…”

Identification of adducin-binding residues on the cytoplasmic domain of erythrocyte membrane protein, band 3

“…However, the observation that the truncated kidney cdb3 binds adducin was surprising in view of the fact that kidney cdb3 displays no known affinity for any other protein with which erythrocyte cdb3 normally interacts, including ankyrin, protein 4.1, glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, and deoxyhemoglobin [28, 39, 46, 47]. Although the interactions of kidney band 3 have received limited study, established protein ligands of kidney cdb3 include integrin-linked kinase, nephrin, and the β1 subunit of the Na,K-ATPase [60–62]. While we were not able to find any literature demonstrating a direct band 3-adducin interaction in the kidney, several studies suggest that mutations in adducin can lead to alterations in levels of nephrin, Na,K-ATPase activity, renal sodium retention, and blood pressure [20–22, 63–65].…”