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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
.
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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 (0.5âng wt-AQP2, R187C, 1.5âng V24A and 2.5âng D150E) and incubated for 24âhours prior testing for water permeability (A) and Western blot (B) on both total and plasma membranes. Water permeability values (Pf) are in %â±âSD of wt-AQP2 with nâ=â8 per condition, and is typical of 5 assays. Asterisks indicate statistical significance in comparison to both water-injected (pâ<â0.001) and wt-AQP2 (pâ=â0.05) oocytes.
Figure 2. Immunoprecipitation of AQP2 in homozygous and heterozygous condition.Oocytes were injected with 0.5âng mRNAs for each untagged AQP2 variants (untagged-WT, V24A, D150E and R187C) along equimolar amounts (1âng) of corresponding GFP-tagged forms (GFP-tagged-WT, V24A, D150E and R187C) and incubated for 24âhours prior immunoprecipitation (IP) using anti-GFP (see methods). Blots were probed using anti-AQP2 and figure shows the pulled-down untagged variants (29âkDa) 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 (pâ=â0.05) in comparison to correspondingâ+âWT conditions.
Figure 3. Functional recovery analysis for rec-AQP2 forms in oocytes.Oocytes were injected with 0.5âng mRNA coding for wt, V24A, D150E or R187C either in absence (â) or presence (+) of same amount of wt-AQP2 and incubated for 24âhours prior testing for water permeability (Pf). Activities are presented in %â±âSD of wt-AQP2 with nâ=â8 per condition, and is typical of 6 assays. (A) Total activity. (B) Specific activity for single (control value subtracted from all (â) 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 (â) to those determined in presence (+) of wt-AQP2. Western blots in panel B represent specific labeling (29âkDa) for each mutant either in absence (â) or presence (+) of GFP-wt-AQP2 (58âkDa, not seen in blot), for both total and plasma membranes. Asterisks indicate statistical significance from zero (pâ=â0.001).
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-AQP2â=â1 to 4). (B) Transposition of theoretical data, plotting activity levels against wt/mut ratios for each minimal requirement (nâ=â1 (â ), 2 (â¡), 3 (â) or 4 (â)). Dash line represent non-interacting condition. C) Oocytes were injected with 1âng mRNA mixtures of varying wt/mut ratios (4/0 to 0/4) combining wtâAQP2 to R254Q, D150E or R187C and incubated for 24âhours prior testing for water permeability. Activities (Pf) are presented in %â±âSD of pure wt-AQP2 with nâ=â8 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 0.5âng mRNA coding for wt, L22V, V24A, A47V, T126M, A147T, D150E, N68S, R187C, V194I or K228E either in absence (â) or presence (+) of same amount of wt-AQP2 and incubated for 24âhours prior testing for water permeability (see Fig. 3). Activities (Pf) are presented in %â±âSD of wt-AQP2 with nâ=â3 to 8 per condition, combining 6 assays. (A) Location of the mutations within the reported structure of AQP2 (33) (redâ=âinterunit faces, blueâ=âexofacial faces). (B) Histogram presenting gain of functionâ±âSD 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 (âWT) 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 (â) 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 meanâ±âSD of cell count in a 60x fieldâ+â8 adjacent fields, for 3 assays. Asterisks indicate statistical significance from -WT condition. (pâ<â0.001).
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