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

2006 ◽  
Vol 291 (6) ◽  
pp. F1354-F1361 ◽  
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.

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

2008 ◽  
Vol 410 (2) ◽  
pp. 271-281 ◽  
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.

scholarly journals Trafficking defects of the Southeast Asian ovalocytosis deletion mutant of anion exchanger 1 membrane proteins

2005 ◽  
Vol 392 (3) ◽  
pp. 425-434 ◽  
Author(s):  
Joanne C. Cheung ◽  
Emmanuelle Cordat ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Cell Surface ◽  
Anion Exchanger ◽  
Mdck Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intercalated Cells ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Human AE1 (anion exchanger 1) is a membrane glycoprotein found in erythrocytes and as a truncated form (kAE1) in the BLM (basolateral membrane) of α-intercalated cells of the distal nephron, where they carry out electroneutral chloride/bicarbonate exchange. SAO (Southeast Asian ovalocytosis) is a dominant inherited haematological condition arising from deletion of Ala400–Ala408 in AE1, resulting in a misfolded and transport-inactive protein present in the ovalocyte membrane. Heterozygotes with SAO are able to acidify their urine, without symptoms of dRTA (distal renal tubular acidosis) that can be associated with mutations in kAE1. We examined the effect of the SAO deletion on stability and trafficking of AE1 and kAE1 in transfected HEK-293 (human embryonic kidney) cells and kAE1 in MDCK (Madin–Darby canine kidney) epithelial cells. In HEK-293 cells, expression levels and stabilities of SAO proteins were significantly reduced, and no mutant protein was detected at the cell surface. The intracellular retention of AE1 SAO in transfected HEK-293 cells suggests that erythroid-specific factors lacking in HEK-293 cells may be required for cell-surface expression. Although misfolded, SAO proteins could form heterodimers with the normal proteins, as well as homodimers. In MDCK cells, kAE1 was localized to the cell surface or the BLM after polarization, while kAE1 SAO was retained intracellularly. When kAE1 SAO was co-expressed with kAE1 in MDCK cells, kAE1 SAO was largely retained intracellularly; however, it also co-localized with kAE1 at the cell surface. We propose that, in the kidney of heterozygous SAO patients, dimers of kAE1 and heterodimers of kAE1 SAO and kAE1 traffic to the BLM of α-intercalated cells, while homodimers of kAE1 SAO are retained in the endoplasmic reticulum and are rapidly degraded. This results in sufficient cell-surface expression of kAE1 to maintain adequate bicarbonate reabsorption and proton secretion without dRTA.

scholarly journals Functional rescue of vasopressin V2 receptor mutants in MDCK cells by pharmacochaperones: relevance to therapy of nephrogenic diabetes insipidus

2007 ◽  
Vol 292 (1) ◽  
pp. F253-F260 ◽  
Author(s):  
J. H. Robben ◽  
M. Sze ◽  
N. V. A. M. Knoers ◽  
P. M. T. Deen
Keyword(s):  
Diabetes Insipidus ◽  
Nephrogenic Diabetes Insipidus ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
High Affinity ◽  
Vasopressin V2 Receptor ◽  
Camp Generation ◽  
V2 Receptor

Intracellular retention of a functional vasopressin V2 receptor (V2R) is a major cause of congenital nephrogenic diabetes insipidus (NDI) and rescue of V2R mutants by nonpeptide antagonists may restore their basolateral membrane (BM) localization and function. However, the criteria for efficient functional rescue of G protein-coupled receptor (GPCR) mutants at clinically feasible antagonist concentrations are unknown. We found that the four nonpeptide antagonists SR49059, OPC31260 , OPC41061 , and SR121463B induced maturation and rescued the BM expression of eight of nine different V2R mutants, stably expressed in physiologically relevant polarized cells. The extent of maturation and rescued BM expression correlated with the antagonists' concentration and affinity for the V2R. Displacement of the antagonists by AVP and subsequent cAMP generation inversely correlated with the antagonists' affinities for the V2R but is partially influenced by antagonist-specific aspects. Despite limited increases in maturation and cell-surface expression of V2R mutants, the low-affinity SR49059 optimally induced functional rescue at high concentrations, due to its easy displacement by vasopressin. At clinically feasible antagonist concentrations, however, only the high-affinity antagonists OPC31260 and OPC41061 induced functional rescue, as at these concentrations the extent of BM expression became limited. In conclusion, functional rescue of mutant V2Rs at clinically feasible concentrations is most effective with high-affinity antagonists. As OPC31260 and OPC41061 are clinically safe, they are promising candidates to relieve NDI. Moreover, as numerous other diseases are caused by endoplasmic reticulum-retained GPCRs for which cell-permeable antagonists become available, our finding that high-affinity antagonists are superior is anticipated to be important for pharmacotherapy development of these diseases.

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

2002 ◽  
Vol 282 (5) ◽  
pp. F810-F820 ◽  
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.

Apical membrane and intracellular distribution of endogenous alpha 2A-adrenergic receptors in MDCK cells

1994 ◽  
Vol 267 (3) ◽  
pp. F347-F353 ◽  
Author(s):  
M. D. Okusa ◽  
K. R. Lynch ◽  
D. L. Rosin ◽  
L. Huang ◽  
Y. Y. Wei
Keyword(s):  
Cell Surface ◽  
De Novo ◽  
Mdck Cells ◽  
Specific Binding ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Surface Binding ◽  
Binding Studies ◽  
Apical Domain

The purpose of the current studies was to characterize the endogenous alpha 2-adrenergic receptor (AR) subtypes present in Madin-Darby canine kidney (MDCK) cells and to determine their level of expression and pattern of distribution. By saturation binding analysis with [3H]MK-912, MDCK cells expressed high levels of alpha 2-ARs with a maximum receptor density (Bmax) of 798 +/- 55 fmol/mg protein and an equilibrium dissociation constant (Kd) of 0.98 +/- 0.32 nM. Competitive binding studies using prazosin, oxymetazoline, phentolamine, and epinephrine to displace [3H]MK-912 demonstrated inhibition constant (Ki) values of 1,270 +/- 250, 5.0 +/- 0.4, 5.5 +/- 0.3, and 392 +/- 150 nM (n = 3), respectively. In Northern blot analysis we found that MDCK cells expressed transcripts encoding alpha 2A-AR and not alpha 2B-AR or alpha 2C-AR. Surface binding experiments suggested that approximately 60% of alpha 2A-ARs are distributed at the cell surface domain. Specific binding of [3H]MK-912 to soluble apical and basolateral surface proteins isolated by surface biotinylation indicated the expression of surface alpha 2A-ARs was limited to the apical domain of MDCK cells. No alpha 2A-ARs were detected on the basolateral surface. We conclude that endogenous alpha 2A-ARs are targeted to the apical domain of MDCK cells and that the intracellular compartment may contain ARs as a reservoir for de novo cell surface expression or, alternatively, may represent internalized receptors.

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

2017 ◽  
Vol 474 (15) ◽  
pp. 2573-2584 ◽  
Author(s):  
Natapol Duangtum ◽  
Mutita Junking ◽  
Suratchanee Phadngam ◽  
Nunghathai Sawasdee ◽  
Andrea Castiglioni ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
Renal Tubular Acidosis ◽  
Mechanistic Explanation ◽  
Distal Renal Tubular Acidosis ◽  
Immunofluorescence Staining ◽  
Cell Surface Expression ◽  
Small Interference Rna ◽  
Golgi Retention ◽  
Renal Tubular

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.

A novel I551F variant of Na+/HCO3- cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity

2021 ◽  
Author(s):  
Osamu Yamazaki ◽  
Maho Yamashita ◽  
Jinping Li ◽  
Fumika Ochiai-Homma ◽  
Tadashi Yoshida ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Surface Expression ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Dominant Negative Effect ◽  
Transport Activity ◽  
Wild Type ◽  
Single Nucleotide Variants

Homozygous mutations in SLC4A4, encoding the electrogenic Na+/HCO3- cotransporter NBCe1, cause proximal renal tubular acidosis (pRTA) associated with extrarenal symptoms. Although 17 mutated sites in SLC4A4 have thus far been identified among pRTA patients, physiological significance of other nonsynonymous single nucleotide variants (SNVs) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From NCBI dbSNP database, we identified 13 SNVs that have not previously been characterized in highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that I551F variant was present predominantly in the cytoplasm in HEK293 cells, whereas all other SNVs did not show as dramatic a change in subcellular distribution. Western blot analysis in HEK293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69 % reduction in cell surface levels. To determine the role of I551 in more detail, we examined the significance of various artificial mutants both in non-polarized HEK293 cells and polarized MDCK cells, which indicated that only I551F substitution resulted in cytoplasmic retention. Moreover, functional analysis using Xenopus oocytes demonstrated that the I551F variant had a significantly reduced activity corresponding to 39 % of that of wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence study in HEK293 cells indicated that the I551F variant retains wild-type NBCe1-A in the cytoplasm. These data demonstrate that I551F-NBCe1-A shows impaired transport activity predominantly through cytoplasmic retention, and suggest that the variant can have a dominant-negative effect by forming complexes with wild-type NBCe1-A.

scholarly journals Short Term Effect of Aldosterone on Na,K-ATPase Cell Surface Expression in Kidney Collecting Duct Cells

2001 ◽  
Vol 276 (50) ◽  
pp. 47087-47093 ◽  
Author(s):  
Vanessa Summa ◽  
David Mordasini ◽  
Frank Roger ◽  
Marcelle Bens ◽  
Pierre-Yves Martin ◽  
...  
Keyword(s):  
Cell Surface ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Extracellular Volume ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Cortical Collecting Duct ◽  
Short Term

Aldosterone controls extracellular volume and blood pressure by regulating Na+reabsorption, in particular by epithelia of the distal nephron. A main regulatory site of this transcellular transport is the epithelial sodium channel (ENaC) that mediates luminal Na+influx. The Na,K-ATPase (Na+pump) that coordinately extrudes Na+across the basolateral membrane is known to be regulated by short term aldosterone as well. We now show that in the cortical collecting duct (CCD) from adrenalectomized rats, the increase in Na,K-ATPase activity (approximately 3-fold in 3 h), induced by a single aldosterone injection, can be fully accounted by the increase in Na,K-ATPase cell surface expression (+ 497 ± 35%). The short term aldosterone action was further investigated in cultured mouse collecting duct principal cells mpkCCDcl4. Within 2 h, maximal Na,K-ATPase function assessed by Na+pump current (Ip) measurements and Na,K-ATPase cell surface expression were increased by 20–50%. Aldosterone did not modify the Na+dependence of the Na+pumps and induced transcription- and translation-dependent actions on pump surface expression and current independently of ENaC-mediated Na+influx. In summary, short term aldosterone directly increases the cell surface expression of pre-existing Na+pumps in kidney CCD target cells. Thus, aldosterone controls Na+reabsorption in the short term not only by regulating the apical cell surface expression of ENaC (Loffing, J., Zecevic, M., Feraille, E., Kaissling, B., Asher, C., Rossier, B. C., Firestone, G. L., Pearce, D., and Verrey, F. (2001)Am. J. Physiol.280, F675–F682) but also by coordinately acting on the basolateral cell surface expression of the Na,K-ATPase.

The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia

2006 ◽  
Vol 291 (4) ◽  
pp. C788-C801 ◽  
Author(s):  
Ashley M. Toye ◽  
Mark D. Parker ◽  
Christopher M. Daly ◽  
Jing Lu ◽  
Leila V. Virkki ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Renal Tubular Acidosis ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Basolateral Membrane ◽  
Surface Expression ◽  
Normal Activity ◽  
Proximal Renal Tubular Acidosis ◽  
Renal Tubular ◽  
The Individual

The human electrogenic renal Na-HCO3cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO3−from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na+affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules.

scholarly journals Regulation of Kir4.1 and AQP4 expression and stability at the basolateral domain of epithelial MDCK cells by the extracellular matrix

2011 ◽  
Vol 301 (2) ◽  
pp. F396-F409 ◽  
Author(s):  
Daniel Kai Long Tham ◽  
Hakima Moukhles
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Surface Expression ◽  
Laminin Receptor ◽  
Cell Surface Expression ◽  
Αvβ3 Integrin ◽  
Diffusion Rates ◽  
Laminin 1

The proper targeting of ion channels to specialized domains is crucial for cell function. Kir4.1, the inwardly rectifying potassium channel, and aquaporin-4 (AQP4), the type 4 water-permeable channel, are localized at the basolateral domain of polarized epithelial cells; however, the mechanisms involved in their localization have yet to be determined. In this study, we investigated the role of the extracellular matrix in the localization of these channels in polarized Madin-Darby canine kidney (MDCK) cells. MDCK cells expressing green fluorescent protein-Kir4.1 or -AQP4 were cultured on laminin-1 or fibronectin and examined by confocal microscopy and cell surface biotinylation to assess plasma membrane expression of Kir4.1 and AQP4. Our data show that laminin-1 and fibronectin induce a significant increase in cell surface expression of both channels at the basolateral domain. Using fluorescence recovery after photobleaching, we demonstrate that laminin-1 and fibronectin reduce the diffusion rates of these channels. Finally, we show that the laminin receptor dystroglycan is important for cell surface expression of Kir4.1 but not AQP4. However, laminin-1 increases cell surface expression of both channels in cells deficient for dystroglycan, indicating that other receptors are involved. Indeed, RGD-containing peptides, which inhibit fibronectin binding to certain integrins, prevent the fibronectin-induced increase in Kir4.1 and AQP4 cell surface expression and reverse the laminin- and fibronectin-induced reduction in both channels' diffusion rates. Similarly, the αvβ3-integrin function-blocking antibody alters the reduction of AQP4 diffusion rates induced by both laminin and fibronectin, suggesting that αvβ3-integrin plays a role in the stabilization of APQ4 at the basolateral domain of epithelial cells.

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