Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Functional Recovery of AQP2 Recessive Mutations Through Hetero-Oligomerization with Wild-Type Counterpart.
El Tarazi A
,
Lussier Y
,
Da Cal S
,
Bissonnette P
,
Bichet DG
.
???displayArticle.abstract???
Aquaporin-2 (AQP2) is a homotetrameric water channel responsible for the final water reuptake in the kidney. Mutations in the protein induce nephrogenic diabetes insipidus (NDI), which challenges the water balance by producing large urinary volumes. Although recessive AQP2 mutations are believed to generate non-functional and monomeric proteins, the literature identifies several mild mutations which suggest the existence of mixed wt/mut tetramers likely to carry function in heterozygotes. Using Xenopus oocytes, we tested this hypothesis and found that mild mutants (V24A, D150E) can associate with wt-AQP2 in mixed heteromers, providing clear functional gain in the process (62 ± 17% and 63 ± 17% increases, respectively), conversely to the strong monomeric R187C mutant which fails to associate with wt-AQP2. In kidney cells, both V24A and D150E display restored targeting while R187C remains in intracellular stores. Using a collection of mutations to expand recovery analyses, we demonstrate that inter-unit contacts are central to this recovery process. These results not only present the ground data for the functional recovery of recessive AQP2 mutants through heteromerization, which prompt to revisit the accepted NDI model, but more importantly describe a general recovery process that could impact on all multimeric systems where recessive mutations are found.
Figure 1. Comparative expression of AQP2 variants in oocytes.Oocytes were injected with mRNAs so to generate equivalent protein loads for each AQP2 variants (0.5 ng wt-AQP2, R187C, 1.5 ng V24A and 2.5 ng D150E) and incubated for 24 hours prior testing for water permeability (A) and Western blot (B) on both total and plasma membranes. Water permeability values (Pf) are in % ± SD of wt-AQP2 with n = 8 per condition, and is typical of 5 assays. Asterisks indicate statistical significance in comparison to both water-injected (p < 0.001) and wt-AQP2 (p = 0.05) oocytes.
Figure 2. Immunoprecipitation of AQP2 in homozygous and heterozygous condition.Oocytes were injected with 0.5 ng mRNAs for each untagged AQP2 variants (untagged-WT, V24A, D150E and R187C) along equimolar amounts (1 ng) of corresponding GFP-tagged forms (GFP-tagged-WT, V24A, D150E and R187C) and incubated for 24 hours prior immunoprecipitation (IP) using anti-GFP (see methods). Blots were probed using anti-AQP2 and figure shows the pulled-down untagged variants (29 kDa) for every IP condition. Results are typical of 4 assays from which individual densitometry were performed and transposed in bar graph as % of wt-AQP2. Asterisks indicate statistical significance (p = 0.05) in comparison to corresponding + WT conditions.
Figure 3. Functional recovery analysis for rec-AQP2 forms in oocytes.Oocytes were injected with 0.5 ng mRNA coding for wt, V24A, D150E or R187C either in absence (−) or presence (+) of same amount of wt-AQP2 and incubated for 24 hours prior testing for water permeability (Pf). Activities are presented in % ± SD of wt-AQP2 with n = 8 per condition, and is typical of 6 assays. (A) Total activity. (B) Specific activity for single (control value subtracted from all (−) expressions), and double (wt-AQP2 value subtracted from all (+) expressions) expressing conditions. (C) Gain of function for each AQP2 variant calculated by subtracting specific Pf values determined in absence (−) to those determined in presence (+) of wt-AQP2. Western blots in panel B represent specific labeling (29 kDa) for each mutant either in absence (−) or presence (+) of GFP-wt-AQP2 (58 kDa, not seen in blot), for both total and plasma membranes. Asterisks indicate statistical significance from zero (p = 0.001).
Figure 4. Functional recovery analysis in function of varying wt/mut ratios.(A) Theoretical model describing activity projections (% pure wt-AQP2) for varying wt/mut ratios (4/0 to 0/4) when assuming a minimal number of functional units within the tetramer to allow activity (n wt-AQP2 = 1 to 4). (B) Transposition of theoretical data, plotting activity levels against wt/mut ratios for each minimal requirement (n = 1 (■), 2 (□), 3 (●) or 4 (○)). Dash line represent non-interacting condition. C) Oocytes were injected with 1 ng mRNA mixtures of varying wt/mut ratios (4/0 to 0/4) combining wt–AQP2 to R254Q, D150E or R187C and incubated for 24 hours prior testing for water permeability. Activities (Pf) are presented in % ± SD of pure wt-AQP2 with n = 8 per condition and is typical of 3 assays. As shown, R254Q display typical dominant negative effect while D150E is compatible with strong functional recovery. As expected, R187C does not affect wt-AQP2 function, in accordance with its non-interfering (monomeric) nature.
Figure 5. Functional recovery of known rec-AQP2 located in distinct areas of the protein.Oocytes were injected with 0.5 ng mRNA coding for wt, L22V, V24A, A47V, T126M, A147T, D150E, N68S, R187C, V194I or K228E either in absence (−) or presence (+) of same amount of wt-AQP2 and incubated for 24 hours prior testing for water permeability (see Fig. 3). Activities (Pf) are presented in % ± SD of wt-AQP2 with n = 3 to 8 per condition, combining 6 assays. (A) Location of the mutations within the reported structure of AQP2 (33) (red = interunit faces, blue = exofacial faces). (B) Histogram presenting gain of function ± SD for each AQP2 variant, as in Fig. 3C. (C) Table presenting specific activities (Pf) along Western bands densitometry (Western) for each mutant in both absence (−WT) and presence (+WT) of wt-AQP2.
Figure 6. Functional recovery of rec-AQP2 forms in mpkCCDc14 cells.Cells grown on semipermeable filters were transfected with pBi vectors expressing FLAG-tagged AQP2 variants (V24A, D150E and R187C) as unique transcript (−) or along untagged wt-AQP2 (+) as second transcript (see methods). Immunofluorescence using anti-FLAG allowed visualization of tagged variants to evaluate effective apical membrane targeting. (A) Induction of plasma membrane targeting for V24A and D150E, but not R187C, by coexpression with wt-AQP2. (B) Histogram of positive apical membrane targeting for each condition, presenting mean ± SD of cell count in a 60x field + 8 adjacent fields, for 3 assays. Asterisks indicate statistical significance from -WT condition. (p < 0.001).
Bissonnette,
Functional expression of tagged human Na+-glucose cotransporter in Xenopus laevis oocytes.
1999, Pubmed,
Xenbase
Bissonnette,
Functional expression of tagged human Na+-glucose cotransporter in Xenopus laevis oocytes.
1999,
Pubmed
,
Xenbase
Boccalandro,
Characterization of an aquaporin-2 water channel gene mutation causing partial nephrogenic diabetes insipidus in a Mexican family: evidence of increased frequency of the mutation in the town of origin.
2004,
Pubmed
Canfield,
Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response.
1997,
Pubmed
,
Xenbase
Christensen,
Localization and regulation of PKA-phosphorylated AQP2 in response to V(2)-receptor agonist/antagonist treatment.
2000,
Pubmed
Deen,
Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing.
1995,
Pubmed
,
Xenbase
Duquette,
Local osmotic gradients drive the water flux associated with Na(+)/glucose cotransport.
2001,
Pubmed
,
Xenbase
Goji,
Novel mutations in aquaporin-2 gene in female siblings with nephrogenic diabetes insipidus: evidence of disrupted water channel function.
1998,
Pubmed
,
Xenbase
Guyon,
Characterization of D150E and G196D aquaporin-2 mutations responsible for nephrogenic diabetes insipidus: importance of a mild phenotype.
2009,
Pubmed
,
Xenbase
Hasler,
Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells.
2002,
Pubmed
Hasler,
Posttranscriptional control of aquaporin-2 abundance by vasopressin in renal collecting duct principal cells.
2006,
Pubmed
Kamsteeg,
Importance of aquaporin-2 expression levels in genotype -phenotype studies in nephrogenic diabetes insipidus.
2000,
Pubmed
,
Xenbase
Kamsteeg,
An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus.
1999,
Pubmed
,
Xenbase
Katsura,
Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.
1995,
Pubmed
Knepper,
Regulation of aquaporin-2 water channel trafficking by vasopressin.
1997,
Pubmed
Kuwahara,
Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus.
2001,
Pubmed
,
Xenbase
Leduc-Nadeau,
Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes.
2007,
Pubmed
,
Xenbase
Leduc-Nadeau,
New autosomal recessive mutations in aquaporin-2 causing nephrogenic diabetes insipidus through deficient targeting display normal expression in Xenopus oocytes.
2010,
Pubmed
,
Xenbase
Marr,
Cell-biologic and functional analyses of five new Aquaporin-2 missense mutations that cause recessive nephrogenic diabetes insipidus.
2002,
Pubmed
,
Xenbase
Marr,
Functionality of aquaporin-2 missense mutants in recessive nephrogenic diabetes insipidus.
2001,
Pubmed
,
Xenbase
Marr,
Heteroligomerization of an Aquaporin-2 mutant with wild-type Aquaporin-2 and their misrouting to late endosomes/lysosomes explains dominant nephrogenic diabetes insipidus.
2002,
Pubmed
Moeller,
Can one Bad Egg' really spoil the batch?
2010,
Pubmed
,
Xenbase
Mulders,
New mutations in the AQP2 gene in nephrogenic diabetes insipidus resulting in functional but misrouted water channels.
1997,
Pubmed
,
Xenbase
Nielsen,
The aquaporin family of water channels in kidney.
1995,
Pubmed
,
Xenbase
Robben,
Cell biological aspects of the vasopressin type-2 receptor and aquaporin 2 water channel in nephrogenic diabetes insipidus.
2006,
Pubmed
Sands,
Nephrogenic diabetes insipidus.
2006,
Pubmed
Savelkoul,
p.R254Q mutation in the aquaporin-2 water channel causing dominant nephrogenic diabetes insipidus is due to a lack of arginine vasopressin-induced phosphorylation.
2009,
Pubmed
Tamarappoo,
Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones.
1998,
Pubmed
,
Xenbase
de Mattia,
Lack of arginine vasopressin-induced phosphorylation of aquaporin-2 mutant AQP2-R254L explains dominant nephrogenic diabetes insipidus.
2005,
Pubmed
de Mattia,
A novel mechanism in recessive nephrogenic diabetes insipidus: wild-type aquaporin-2 rescues the apical membrane expression of intracellularly retained AQP2-P262L.
2004,
Pubmed
,
Xenbase
