X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking

Abstract

Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd 2+ -ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.membrane protein | X-ray crystallography | water channel protein W ater is the major ingredient of the human body, constituting 55-65% of our total body weight (1). Water homeostasis is maintained by the kidneys, which filter ∼180 L of primary urine every day. Although most water is constitutively reabsorbed in the proximal tubules and descending limbs of Henle (2), the body's water balance is fine-tuned by regulated water reabsorption, which takes place in the kidney collecting duct. Water reabsorption is mediated by aquaporins, membranebound water channels, of which seven of the 13 human isoforms have been located in the human kidney (3). Of these, human aquaporin 2 (AQP2) is present in the principal cells of the collecting duct and is responsible for regulated water reabsorption.AQP2 is stored in intracellular vesicles under water-saturating conditions. When the levels of the pituitary antidiuretic hormone arginine vasopressin (AVP) are elevated in response to dehydration or hypernatremia, AVP binding to the vasopressin 2 receptor (V2R) in the basolateral membrane stimulates an increase in intracellular cAMP. This triggers the phosphorylation of Ser256 in the AQP2 C terminus by protein kinase A (PKA) and flags the protein for trafficking from storage vesicles to the apical membrane (4-6). AVP also triggers additional phosphorylation at Ser264 and Ser269 (7,8), with all three sites being phosphorylated in AQP2s targeted to the plasma membrane (9). The resulting redistribution of AQP2 increases transcellular water permeability and concentrates urine (Fig. S1). Once correct water balance is restored, AQP2 is internalized through ubiquitinmediated endocytosis and redirected to storage vesicles or targeted for degradation (10-12).Because of its central role in water homeostasis, dysregula...

Pathophysiology, diagnosis and management of nephrogenic diabetes insipidus

“…Dominant mutations typically affect amino acids in the C-terminus, leading to aberrant trafficking; mutant AQP2 subunits are able to oligomerize with wildtype protein to form tetramers, but exert a dominant-negative effect on trafficking of these assembled tetramers. 52 Interestingly, specific mutations direct AQP2 trafficking to distinct cellular compartments, such as the Golgi complex, 13 late endosomes and lysosomes 49 or the basolateral membrane. 51,53 These findings have provided insights into the intracellular trafficking motifs of AQP2.…”

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

“…18,19 The AQP0-construct contained a C-terminal 6 Â His-tag whereas the AQP2-construct contained an N-terminal 8 Â His-tag. Cells were grown in a fermenter (Belach Bioteknik) and protein expression was induced using methanol for 24-36 hours for AQP2 and 120 hours for AQP0, resulting in a typical yield of 300 g wet cells per litre of culture.…”

Beyond water homeostasis: Diverse functional roles of mammalian aquaporins

“…This is because the majority of AQP crystal structures are obtained using constructs in which the N-and C-terminal tails of the protein have been truncated due the difficulty of obtaining well-diffracting crystals using fulllength AQP molecules [41]. This suggests an inherent structural flexibility in these regions of AQP molecules and indeed a recent X-ray structure of AQP2, which included ~20 residues of the (truncated) C-terminal tail, found this fragment of the Cterminus in four strikingly different conformations in each of the four AQP2 monomers within the tetrameric unit cell [42].…”