γ-COPI mediates the retention of kAE1 G701D protein in Golgi apparatus – a mechanistic explanation of distal renal tubular acidosis associated with the G701D mutation

Mutations of the solute carrier family 4 member 1 (SLC4A1) gene encoding kidney anion (chloride/bicarbonate ion) exchanger 1 (kAE1) can cause genetic distal renal tubular acidosis (dRTA). Different SLC4A1 mutations give rise to mutant kAE1 proteins with distinct defects in protein trafficking. The mutant kAE1 protein may be retained in endoplasmic reticulum (ER) or Golgi apparatus, or mis-targeted to the apical membrane, failing to display its function at the baso-lateral membrane. The ER-retained mutant kAE1 interacts with calnexin chaperone protein; disruption of this interaction permits the mutant kAE1 to reach the cell surface and display anion exchange activity. However, the mechanism of Golgi retention of mutant kAE1 G701D protein, which is otherwise functional, is still unclear. In the present study, we show that Golgi retention of kAE1 G701D is due to a stable interaction with the Golgi-resident protein, coat protein complex I (COPI), that plays a role in retrograde vesicular trafficking and Golgi-based quality control. The interaction and co-localization of kAE1 G701D with the γ-COPI subunit were demonstrated in human embryonic kidney (HEK-293T) cells by co-immunoprecipitation and immunofluorescence staining. Small interference RNA (siRNA) silencing of COPI expression in the transfected HEK-293T cells increased the cell surface expression of transgenic kAE1 G701D, as shown by immunofluorescence staining. Our data unveil the molecular mechanism of Golgi retention of kAE1 G701D and suggest that disruption of the COPI-kAE1 G701D interaction could be a therapeutic strategy to treat dRTA caused by this mutant.

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Impaired trafficking of distal renal tubular acidosis mutants of the human kidney anion exchanger kAE1

AJP Renal Physiology ◽

10.1152/ajprenal.00216.2001 ◽

2002 ◽

Vol 282 (5) ◽

pp. F810-F820 ◽

Cited By ~ 55

Author(s):

Janne A. Quilty ◽

Jing Li ◽

Reinhart A. Reithmeier

Keyword(s):

Cell Surface ◽

Renal Tubular Acidosis ◽

Anion Exchanger ◽

Human Kidney ◽

Distal Renal Tubular Acidosis ◽

Dominant Negative ◽

Cell Surface Expression ◽

Dominant Negative Effect ◽

Inherited Disease ◽

Renal Tubular

Distal renal tubular acidosis (dRTA) is an inherited disease characterized by the failure of the kidneys to appropriately acidify urine and is associated with mutations in the anion exchanger (AE)1 gene. The effect of the R589H dRTA mutation on the expression of the human erythroid AE1 and the truncated kidney form (kAE1) was examined in transfected human embryonic kidney 293 cells. AE1, AE1 R589H, and kAE1 were present at the cell surface, whereas kAE1 R589H was located primarily intracellularly as shown by immunofluorescence, cell surface biotinylation, N-glycosylation, and anion transport assays. Coexpression of kAE1 R589H reduced the cell surface expression of kAE1 and AE1 by a dominant-negative effect, due to heterodimer formation. The mutant AE1 and kAE1 bound to an inhibitor affinity resin, suggesting that they were not grossly misfolded. Other mutations at R589 also prevented the formation of the cell surface form of kAE1, indicating that this conserved arginine residue is important for proper trafficking. The R589H dRTA mutation creates a severe trafficking defect in kAE1 but not in erythroid AE1.

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Dominant-negative effect of Southeast Asian ovalocytosis anion exchanger 1 in compound heterozygous distal renal tubular acidosis

Biochemical Journal ◽

10.1042/bj20070615 ◽

2008 ◽

Vol 410 (2) ◽

pp. 271-281 ◽

Cited By ~ 11

Author(s):

Saranya Kittanakom ◽

Emmanuelle Cordat ◽

Reinhart A. F. Reithmeier

Keyword(s):

Cell Surface ◽

Blood Cell ◽

Renal Tubular Acidosis ◽

Mdck Cells ◽

Basolateral Membrane ◽

Surface Expression ◽

Cell Surface Expression ◽

Compound Heterozygous ◽

Intercalated Cells ◽

Renal Tubular

The human chloride/bicarbonate AE1 (anion exchanger) is a dimeric glycoprotein expressed in the red blood cell membrane, and expressed as an N-terminal (Δ1–65) truncated form, kAE1 (kidney AE1), in the basolateral membrane of α-intercalated cells in the distal nephron. Mutations in AE1 can cause SAO (Southeast Asian ovalocytosis) or dRTA (distal renal tubular acidosis), an inherited kidney disease resulting in impaired acid secretion. The dominant SAO mutation (Δ400–408) that results in an inactive transporter and altered eythrocyte shape occurs in many dRTA families, but does not itself result in dRTA. Compound heterozygotes of four dRTA mutations (R602H, G701D, ΔV850 and A858D) with SAO exhibit dRTA and abnormal red blood cell properties. Co-expression of kAE1 and kAE1 SAO with the dRTA mutants was studied in polarized epithelial MDCK (Madin–Darby canine kidney) cells. Like SAO, the G701D and ΔV850 mutants were predominantly retained intracellularly, whereas the R602H and A858D mutants could traffic to the basolateral membrane. When co-expressed in transfected cells, kAE1 WT (wild-type) and kAE1 SAO could interact with the dRTA mutants. MDCK cells co-expressing kAE1 SAO with kAE1 WT, kAE1 R602H or kAE1 A858D showed a decrease in cell-surface expression of the co-expressed proteins. When co-expressed, kAE1 WT co-localized with the kAE1 R602H, kAE1 G701D, kAE1 ΔV850 and kAE1 A858D mutants at the basolateral membrane, whereas kAE1 SAO co-localized with kAE1 WT, kAE1 R602H, kAE1 G701D, kAE1 ΔV850 and kAE1 A858D in MDCK cells. The decrease in cell-surface expression of the dRTA mutants as a result of the interaction with kAE1 SAO would account for the impaired expression of functional kAE1 at the basolateral membrane of α-intercalated cells, resulting in dRTA in compound heterozygous patients.

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Expression and interaction of two compound heterozygous distal renal tubular acidosis mutants of kidney anion exchanger 1 in epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00015.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. F1354-F1361 ◽

Cited By ~ 20

Author(s):

Emmanuelle Cordat ◽

Reinhart A. F. Reithmeier

Keyword(s):

Cell Surface ◽

Renal Tubular Acidosis ◽

Mdck Cells ◽

Basolateral Membrane ◽

Surface Expression ◽

Cell Surface Expression ◽

Compound Heterozygous ◽

Intercalated Cells ◽

Affinity Resin ◽

Renal Tubular

Kidney AE1 (kAE1) is a glycoprotein responsible for the electroneutral exchange of chloride for bicarbonate, promoting the reabsorption of bicarbonate into the blood by α-intercalated cells of the collecting tubule. Mutations occurring in the gene encoding kAE1 can induce defects in urinary acidification resulting in distal renal tubular acidosis (dRTA). We expressed two kAE1 dRTA mutants, A858D, a mild dominant mutation, and ΔV850, a recessive mutation, in epithelial Madin-Darby canine kidney (MDCK) cells. Individuals heterozygous with wild-type (WT) kAE1 either did not display any symptoms of dRTA (ΔV850/WT) or displayed a mild incomplete form of dRTA (A858D/WT), while compound heterozygotes (ΔV850/A858D) had dRTA. We found that the A858D mutant was slightly impaired in the endoplasmic reticulum (ER) exit but could target to the basolateral membrane of polarized MDCK cells. Despite an altered binding to an inhibitor affinity resin, anion transport assays showed that the A858D mutant was functional at the cell surface. The ΔV850 mutant showed altered binding to the affinity resin but was predominantly retained in the ER, resulting in undetectable AE1 expression at the basolateral membrane. When coexpressed in MDCK cells, the WT protein, and to a lesser extent the A858D mutant, enhanced the cell surface expression of the ΔV850 mutant. The ΔV850 mutant also affected the cell surface expression of the A858D mutant. Compound heterozygous (A858D/ΔV850) patients likely possess a decreased amount of functional anion exchangers at the basolateral membrane of their α-intercalated cells, resulting in impaired bicarbonate transport into the blood and defective acid transport into the urine.

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Band 3 Walton, a C-terminal deletion associated with distal renal tubular acidosis, is expressed in the red cell membrane but retained internally in kidney cells

Blood ◽

10.1182/blood.v99.1.342 ◽

2002 ◽

Vol 99 (1) ◽

pp. 342-347 ◽

Cited By ~ 56

Author(s):

Ashley M. Toye ◽

Lesley J. Bruce ◽

Robert J. Unwin ◽

Oliver Wrong ◽

Michael J. A. Tanner

Keyword(s):

Cell Surface ◽

Renal Tubular Acidosis ◽

Band 3 ◽

Normal Band ◽

Distal Renal Tubular Acidosis ◽

Mutant Protein ◽

Kidney Cells ◽

Kidney Cell Line ◽

Transport Activity ◽

Renal Tubular

Human band 3 Walton is an AE1 mutation that results in the deletion of the 11 COOH-terminal amino acids of the protein and is associated with dominant distal renal tubular acidosis. The properties of band 3 Walton expressed with normal band 3 in the heterozygous mutant erythrocytes and the kidney isoform expressed in Xenopusoocytes and in the Madin-Darby canine kidney cell line were examined. The mutant erythrocytes have normal hematology but have reduced band 3 Walton content. Transport studies showed that erythrocyte band 3 Walton has normal sulfate transport activity, and kidney band 3 Walton has normal chloride transport activity when expressed inXenopus oocytes. The mutant protein is clearly able to reach the cell surface of erythrocytes and oocytes. In contrast, while normal kidney band 3 was expressed at the cell surface in the kidney cell line, the Walton mutant protein was retained intracellularly within the kidney cells. The results demonstrate that band 3 Walton is targeted differently in erythrocytes and kidney cells and indicate that the COOH-terminal tail of band 3 is required to allow movement to the cell surface in kidney cells. It is proposed here that the mutant band 3 gives rise to dominant distal renal tubular acidosis by inhibiting the movement of normal band 3 to the cell surface. It is suggested that this results from the association of the normal and mutant proteins in band 3 hetero-oligomers, which causes the intracellular retention of normal band 3 with the mutant protein.

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