Functional Recovery of AQP2 Recessive Mutations Through Hetero-Oligomerization with Wild-Type Counterpart

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.

Fungal X-Intrinsic Protein Aquaporin from Trichoderma atroviride: Structural and Functional Considerations

“…All these results increase the sense of plausible interconnected compensating effects between Triat MIP from a same subgroup and counterparts belonging to the three different subgroups, in particular to facilitating common transport across biomembranes of water and a variety of low molecular weight solutes which need to be identified. As a complement, these compensating events do not exclude the possibility of the divergent function of certain MIP members belonging to each subgroup, but whose combination remains essential for the metabolic integrity of the organism: first by the amount of permeable activity from each protomer, but also for the tetramer structures (homo- and hetero-oligomers) where permeability activity can be potentiated when protomers interact with each other in a cooperative mechanism, as observed for yeast, plant and mammal MIP [ 121 , 122 , 123 , 124 ]. To the best of our knowledge, there is no homo- or hetero-oligomer complex described in filamentous MIP, opening a vast new field to be further explored.…”

Further evidence for functional recovery of AQP2 mutations associated with nephrogenic diabetes insipidus

“…All mutations for expression in oocytes were generated using site‐directed mutagenesis on the pT7Ts‐AQP2 vector, as previously described (Guyon et al, 2009; Leduc‐Nadeau et al, 2010) and constructs were validated through sequencing. For production of pT7Ts‐GFP‐AQP2, refer to (El Tarazi et al, 2016). Vectors were linearized with SalI from NEB, and cRNAs was synthesized using the mMessage mMachine T7 kit (Thermo Fisher Scientific).…”

“…However, our recent work describing rec ‐AQP2 mutations of mild phenotypes not only stressed that such mutations were not actually ill‐structured but most importantly demonstrated their capacity to associate with wt ‐AQP2 within structured channels, recuperating function and proper targeting in the process (El Tarazi et al, 2016; Guyon et al, 2009; Leduc‐Nadeau et al, 2010). This functional recovery of rec ‐AQP2 mutations, first identified with P262L (de Mattia et al, 2004), now shows to be wide spread and thus prompt for the introduction of a new class of recessive mutants and challenges the proposed model for NDI (Kamsteeg et al, 1999; Robben et al, 2006).…”

“…Using Xenopus laevis oocytes, we have performed both functional analysis and biochemical characterization of all mutants, probing both pure and coexpression conditions to determine their basic biochemical features with special emphasis on the possible recovery process through wt ‐AQP2 hetero‐oligomerization, as done previously in our lab (El Tarazi et al, 2016; Guyon et al, 2009; Leduc‐Nadeau et al, 2010). Here again, data conflict with the standing NDI model and show that functionally recovered mutants are not the exception but rather constitute the standard outcome for such mutations, which prompted us in proposing a revised model for NDI.…”

Plant and Mammal Aquaporins: Same but Different

“…Co-immunoprecipitation of mutated AQP2 (763–772del) with WT AQP2 demonstrated their ability to interact and build so-called hetero-oligomers [ 274 ]. Heteromerization of WT AQP2 can reduce the deleterious impact of recessive mutations (recAQP2) responsible for NDI [ 275 ]. recAQP2 is believed to possess an abnormal folding impeding the proper oligomerization and, therefore, preventing its targeting to the plasma membrane or leading to its degradation.…”