Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Nyblom, Maria; Frick, Anna; Wang, Yi; Ekvall, Mikael T.; Hallgren, Karin; Hedfalk, Kristina; Neutze, Richard; Tajkhorshid, Emad; Törnroth-Horsefield, Susanna

doi:10.1016/j.jmb.2009.01.065

Cited by 97 publications

(101 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Molecular dynamic simulations indicate that Ser-115 phosphorylation triggers this movement of loop D (31). Furthermore, x-ray structural analysis of phosphorylation mutants suggests that phosphorylation at Ser-188 may also produce an open channel, which was supported by an increased water transport activity of this mutant and also molecular dynamics simulations (32). These simulations have revealed interactions between phosphorylated Ser-188 and Lys-270 of the C terminus that lead to a conformational change in loop D and channel opening.…”

Section: Discussionmentioning

confidence: 64%

Phosphorylation of Aquaporin-2 Regulates Its Water Permeability

Eto

Noda

Horikawa

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylationmimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.Body water homeostasis is essential for the survival of mammals and is regulated by the renal collecting duct. Key components in the regulation of collecting duct water permeability are the vasopressin receptor and the water channel aquaporin-2 (AQP2) 2 (1-6). Mutations in the vasopressin V 2 receptor and AQP2 cause congenital nephrogenic diabetes insipidus, a disease characterized by a massive loss of water through the kidney (7,8). The binding of the antidiuretic hormone vasopressin to vasopressin V 2 receptors on renal principal cells stimulates cAMP synthesis via activation of adenylate cyclase. The subsequent activation of PKA leads to phosphorylation of AQP2 at Ser-256, and this phosphorylation event is required to increase the water permeability and water reabsorption of renal principal cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. We showed recently that AQP2 binds to a multiprotein "motor" complex and that phosphorylation-dependent reciprocal interaction with G-actin and tropomyosin 5b directs AQP2 to the apical membrane (9 -13). However, whether this phosphorylation regulates the water permeability of individual AQP2 proteins still remains unclear. Kuwahara et al. (14) showed that cAMP stimulation increased the water permeability of AQP2 expressed in Xenopus oocytes, alt...

show abstract

Section: Discussionmentioning

confidence: 64%

Phosphorylation of Aquaporin-2 Regulates Its Water Permeability

Eto

Noda

Horikawa

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…*R free was calculated for 5% of reflections randomly excluded from the refinement. SoPIP2;1, where the extension of TM helix 1 disrupts a Ca 2+ binding site implied in gating (27). The reoccurrence of these two specific N-terminal conformations among regulated eukaryotic aquaporins suggests a structural principle governing their regulation.…”

Section: Resultsmentioning

confidence: 99%

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

Frick

Eriksson

Mattia

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

127

111

View full text Add to dashboard Cite

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...

show abstract

“…Several aquaporin structures have been resolved by x-ray crystallography, including human AQP1, human AQP5, and spinach (Spinacia oleracea) aquaporin PIP2 (Ren et al, 2000;Fujiyoshi et al, 2002;Kukulski et al, 2005;Horsefield et al, 2008;Nyblom et al, 2009). We aligned hAQP1, hAQP5, and So-PIP2 with At-PIP2;1, and based on this model, we speculated that L81, W85, and F210 might play a role in CO 2 permeability.…”

Section: Functional Pip2;1 Is Required For the Extracellular Co 2 Resmentioning

confidence: 93%

Reconstitution of CO₂ Regulation of SLAC1 Anion Channel and Function of CO₂-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor

et al. 2016

View full text Add to dashboard Cite

Dark respiration causes an increase in leaf CO 2 concentration (Ci), and the continuing increases in atmospheric [CO 2 ] further increases Ci. Elevated leaf CO 2 concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO 2 /HCO 3 2 enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the bCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO 2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO 2 -and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of bCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO 2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO 2 permeability and extracellular CO 2 regulation of SLAC1 activity. These findings identify the CO 2 -permeable PIP2;1 as key interactor of bCA4 and demonstrate functional reconstitution of extracellular CO 2 signaling to ion channel regulation upon coexpression of PIP2;1, bCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO 2 regulation of S-type anion channels.

show abstract

Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Cited by 97 publications

References 51 publications

Phosphorylation of Aquaporin-2 Regulates Its Water Permeability

Phosphorylation of Aquaporin-2 Regulates Its Water Permeability

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

Reconstitution of CO₂ Regulation of SLAC1 Anion Channel and Function of CO₂-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor

Contact Info

Product

Resources

About

Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Cited by 97 publications

References 51 publications

Phosphorylation of Aquaporin-2 Regulates Its Water Permeability

Phosphorylation of Aquaporin-2 Regulates Its Water Permeability

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

Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor

Contact Info

Product

Resources

About

Reconstitution of CO₂ Regulation of SLAC1 Anion Channel and Function of CO₂-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor