研究者詳細 - 安井　正人

論文 - 安井　正人

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Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

Kadohira Ikuko, Abe Yoichiro, Nuriya Mutsuo, Sano Kazumi, Tsuji Shoji, Arimitsu Takeshi, Yoshimura Yasunori, Yasui Masato

Biochemical and Biophysical Research Communications 377 （ 2 ） 463 - 468 2008年12月

ISSN 0006-291X

Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [³²P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition. © 2008 Elsevier Inc. All rights reserved.

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[Roles of aquaporins in the central nervous system].

Yasui Masato

Masui. The Japanese journal of anesthesiology 57 Suppl 2008年11月

ISSN 0021-4892

Erratum

Yukutake Yoshinori, Tsuji Shoji, Hirano Yoshinori, Adachi Takeshi, Takahashi Toshiyuki, Fujihara Kazuo, Agre Peter, Yasui Masato, Suematsu Makoto

Biology of the Cell 100 （ 9 ） 561 2008年09月

ISSN 0248-4900

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes

Yukutake Yoshinori, Tsuji Shoji, Hirano Yoshinori, Adachi Takeshi, Takahashi Toshiyuki, Fujihara Kazuo, Agre Peter, Yasui Masato, Suematsu Makoto

Biology of the Cell 100 （ 6 ） 355 - 363 2008年06月

ISSN 0248-4900

Background information. Mercurials inhibit AQPs (aquaporins), and site-directed mutagenesis has identified Cys¹⁸⁹ as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury-insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys¹⁸⁹ of AQP1. Indeed, the osmotic water permeability (P_f) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl_2-. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His-tagged rAPR4 (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni²⁺-nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. Results. The water permeability of AQP4 proteoliposomes with or without HgCl₂ was measured with a stopped-flow apparatus. Surprisingly, the P_f of AQP4 proteoliposomes was significantly decreased by 5 μM HgCl₂ within 30 s, and this effect was completely reversed by 2-mercaptoethanol. The dose- and time-dependent inhibitory effects of Hg²⁺ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site-directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys¹⁷⁸ which is located at loop D facing the intracellular side, is a target responding to Hg²6+. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions. Our results suggest that mercury inhibits the P_f of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.

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The difference of aquaporin 5 distribution in acinar and ductal cells in lacrimal and parotid glands

Sasaki Yasumasa, Tsubota Kazuo, Kawedia Jitesh D., Menon Anil G., Yasui Masato

Current Eye Research 32 （ 11 ） 923 - 929 2007年11月

ISSN 0271-3683

Purpose: This study was designed to clarify the physiological function and tissue distribution of aquaporin 5 (AQP5) in the lacrimal and parotid glands. Methods: Saliva and tear volumes were compared in AQP5 knockout (AQP5-/-) mice and wild-type mice. Immunohistochemistry and immunoblot analysis were performed in wild-type and AQP5-/- mice. Results: Immunofluorescence of AQP5 staining showed that AQP5 was localized mainly in the ductal cells rather than in the acinar cells of the lacrimal gland. In contrast, in the parotid gland, AQP5 was observed abundantly in acinar cells with undetectable staining in ductal cells. Tear secretion was not changed in AQP5-/- mouse, although saliva secretion was significantly reduced. Conclusions: AQP5 distribution in acinar cells and ductal cells was completely opposite in the lacrimal and parotid glands. The physiological role of AQP5 might be dependent on the characteristic tissue distribution of the protein in the lacrimal and parotid glands. Copyright © Informa Healthcare USA, Inc.

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Molecular cloning and characterization of mouse aquaporin 6

Nagase Hiroaki, Ågren Johan, Saito Akiko, Liu Kun, Agre Peter, Hazama Akihiro, Yasui Masato

Biochemical and Biophysical Research Communications 352 （ 1 ） 12 - 16 2007年01月

ISSN 0006-291X

In the rat kidney, aquaporin (AQP) 6 is localized in the intracellular vesicle membranes of type-A intercalated cells of the collecting duct; mouse AQP6 (mAQP6) has not been characterized. Although mAQP6 was originally cloned from cDNA in a mouse cerebellum library (GenBank NM 175087), we have independently cloned a cDNA encoding mAQP6 from an adult kidney cDNA library (C57BL/6J strain). We identified two different spliced variants of mAQP6: mAQP6a and mAQP6b. The mAQP6a isoform is almost identical to that of rat AQP6, whereas mAQP6b is identical to that reported in the mouse cerebellum library mentioned above. We found that the mRNA expression of these two spliced variants is regulated in a tissue-specific and age-dependent manner. Functional analyses of water and ion permeation revealed that mAQP6a functions like rat AQP6 and that mAQP6b does not function as either a water channel or an ion channel under our experimental conditions. © 2006 Elsevier Inc. All rights reserved.

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Determinants of AQP6 trafficking to intracellular sites versus the plasma membrane in transfected mammalian cells

Beitz Eric, Liu Kun, Ikeda Masahiro, Guggino William B., Agre Peter, Yasui Masato

Biology of the Cell 98 （ 2 ） 101 - 109 2006年02月

ISSN 0248-4900

Background information. Most AQPs (aquaporins) function at the plasma membrane, however AQP6 is exclusively localized to membranes of intracellular vesicles in acid-secreting type-A intercalated cells of renal collecting ducts. The intracellular distribution indicates that AQP6 has a function distinct from trans-epithelial water movement. Results. We show by mutational analyses and immunofluorescence that the N-terminus of AQP6 is a determinant for its intracellular localization. Presence or absence at the plasma membrane of AQP6 constructs was confirmed by electrophysiological methods. Addition of a GFP (green fluorescent protein) or a HA (haemagglutinin) epitope tag (GFP-AQP6 or HA-AQP6) to the N-terminus of AQP6, directed AQP6 to the plasma membranes of transfected Madin-Darby canine Kidney cells. In contrast, addition of a GFP tag to the C-terminus (AQP6-GFP) caused the protein to remain intracellular, similar to untagged wild-type AQP6. Replacement of the N-terminus of AQP6 by that of AQP1 also directed AQP6 to the plasma membranes, whereas the N-terminus of AQP6 retained AQP1 in cytosolic sites. Conclusion. Our results suggest that the N-terminus of AQP6 is critical for trafficking of the protein to the intracellular sites. Moreover, our studies provide an approach for future identification of proteins involved in vesicle sorting in the acid-secreting type-A intercalated cells.

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Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M₃ mAChRs in interlobular ducts of rat parotid gland

Ishikawa Yasuko, Yuan Zhenfang, Inoue Noriko, Skowronski Mariusz T., Nakae Yoshiko, Shono Masayuki, Cho Gota, Yasui Masato, Agre Peter, Nielsen Søren

American Journal of Physiology - Cell Physiology 289 （ 5 58-5 ） 2005年11月

ISSN 0363-6143

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M₃ muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M₃ mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca²⁺ signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands. Copyright © 2005 the American Physiological Society.

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Conversion of aquaporin 6 from an anion channel to a water-selective channel by a single amino acid substitution

Liu Kun, Kozono David, Kato Yasuhiro, Agre Peter, Hazama Akihiro, Yasui Masato

Proceedings of the National Academy of Sciences of the United States of America 102 （ 6 ） 2192 - 2197 2005年02月

ISSN 0027-8424

Aquaporin (AQP) 6 belongs to the aquaporin water channel family. Unlike other aquaporins, AQP6 functions not as a water channel but as an anion-selective channel. Single-channel analyses have shown AQP6 to flicker rapidly between closed and open status. The atomic structure of AQP1 and amino acid sequence alignments of the mammalian aquaporins reveal two well conserved glycine residues: Gly-57 in transmembrane helix (TM) 2 and Gly-173 in TM5 reside at the contact point where the two helices cross in human AQP1. Uniquely, all known mammalian orthologs of AQP6 have an asparagine residue (Asn-60) at the position corresponding to Gly-57. Here we show that a single residue substitution (N60G in rat AQP6) totally eliminates the anion permeability of AQP6 when expressed in Xenopus oocytes, but the N60G oocytes exhibit significantly higher osmotic water permeability under basal conditions. Replacement of the glycine at this site in AQP0, AQP1, and AQP2 blocked expression of the mutants at the oocyte plasma membrane. We propose that the asparagine residue at the contact point between TM2 and TM5 in AQP6 may function as a teeter board needed for rapid structural oscillations during anion permeation.

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Molecular mechanisms and drug development in aquaporin water channel diseases

Yasui Masato

Journal of Pharmacological Sciences 96 （ 3 ） 260 - 263 2004年11月

ISSN 1347-8613

The discovery of the water channel aquaporin has greatly expanded our understanding of the regulation of the water permeability of biological membranes. The atomic structure of aquaporin-1 (AQP1) demonstrated how aquaporin is freely permeated by water but not protons and provided marked insight into several human disorders. Eleven mammalian aquaporins have been identified, each with a distinct distribution, and these are selectively permeated by water or water plus glycerol. Aquaporins are suspected in numerous pathological conditions involving fluid transport such as brain edema. Knowledge of aquaporin structure may provide insight into the development of new therapeutics through appropriate drug design.

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Molecular dissection of water and glycerol permeability of the aquaglyceroporin from Plasmodium falciparum by mutational analysis

Beitz Eric, Pavlovic-Djuranovic Slavica, Yasui Masato, Agre Peter, Schultz Joachim E.

Proceedings of the National Academy of Sciences of the United States of America 101 （ 5 ） 1153 - 1158 2004年02月

ISSN 0027-8424

The selectivity of aquaporins for water and solutes is determined by pore diameter. Paradoxically, the wider pores of glycerol facilitators restrict water passage by an unknown mechanism. Earlier we characterized an aquaglyceroporin from Plasmodium falciparum with high permeability for both glycerol and water. We use point mutations to demonstrate that amino acids directly lining the pore are not responsible for the excellent water permeability of the Plasmodium aquaglyceroporin but affect permeability of pentitols. Within a conserved WET triad in the extracellular C-loop we identified a Plasmodium aquaglyceroporin-specific glutamate (E125) located in proximity to a conserved arginine (R196) at the pore mouth. Mutation of E125 to serine largely abolished water permeability. Concomitantly, the activation energy for water permeation was increased by 4 kcal/mol. Mutation of the adjacent tryptophan to cysteine led to irreversible inhibition of water passage by Hg²⁺. This unequivocally proves the proximity of the couple WI24/E125 close to the pore mouth. We conclude that in the Plasmodium aquaglyceroporin the electrostatic environment at the extracellular pore entry regulates water permeability.

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Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63

Ikeda Masahiro, Beitz Eric, Kozono David, Guggino William B., Agre Peter, Yasui Masato

Journal of Biological Chemistry 277 （ 42 ） 39873 - 39879 2002年10月

ISSN 0021-9258

Aquaporins (AQP) were originally regarded as plasma membrane channels that are freely permeated by water or small uncharged solutes but not by ions. Unlike other aquaporins, AQP6 overexpressed in Xenopus laevis oocytes was previously found to exhibit Hg²⁺ or pH-activated ion conductance. AQP6 could not be analyzed electrophysiologically in mammalian cells, however, because the protein is restricted to intracellular vesicles. Here we report that addition of a green fluorescence protein (GFP) tag to the N terminus of rat AQP6 (GFP-AQP6) redirects the protein to the plasma membranes of transfected mammalian cells. This permitted measurement of rapid, reversible, pH.induced anion currents by GFP-AQP6 in human embryonic kidney 293 cells. Surprisingly, anion selectivity relative to Cl^- revealed high nitrate permeability even at pH 7.4; P_NO3/P_Cl >9.8. Site-directed mutation of a pore-lining threonine to isoleucine at position 63 at the midpoint of the channel reduced NO₃
^-/Cl^- selectivity. Moreover, no anomalous mole-fraction behavior was observed with NO₃
^-/Cl^- mixtures, suggesting a single ion-binding pore in each subunit. Our studies indicate that AQP6 exhibits a new form of anion permeation with marked specificity for nitrate conferred by a specific pore-lining residue, observations that imply that the primary role of AQP6 may be in cellular regulation rather than simple fluid transport.

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Aquaporins and disease

King Landon S., Yasui Masato

Trends in Endocrinology and Metabolism 13 （ 8 ） 355 - 360 2002年10月

ISSN 1043-2760

Recent discovery of a family of water-specific membrane channel proteins, the aquaporins, has provided new insights into the molecular basis of membrane water permeability. Eleven mammalian aquaporins have been identified to date, with homolog present across the spectrum of life, including bacteria, yeast and plants. The distribution of the mammalian aquaporins predicts their participation in a range of pathophysiological events. Empirical evidence of a physiological role for aquaporins is emerging from studies in both mice and humans, and suggests that aquaporins are likely to play significant roles in human pathophysiology.

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Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation.

Hazama Akihiro, Kozono David, Guggino William B., Agre Peter, Yasui Masato

Journal of Biological Chemistry 277 （ 32 ） 29224 - 29230 2002年08月

ISSN 0021-9258

Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 microm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 microm HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by 22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel.

Aquaporin water channels - From atomic structure to clinical medicine

Agre Peter, King Landon S., Yasui Masato, Guggino Wm B., Ottersen Ole Petter, Fujiyoshi Yoshinori, Engel Andreas, Nielsen Søren

Journal of Physiology 542 （ 1 ） 3 - 16 2002年07月

ISSN 0022-3751

The water permeability of biological membranes has been a longstanding problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. AQP1 is selectively permeated by water driven by osmotic gradients. The atomic structure of human AQP1 has recently been defined. Each subunit of the tetramer contains an individual aqueous pore that permits single-file passage of water molecules but interrupts the hydrogen bonding needed for passage of protons. At least 10 mammalian aquaporins have been identified, and these are selectively permeated by water (aquaporins) or water plus glycerol (aquaglyceroporins). The sites of expression coincide closely with the clinical phenotypes - ranging from congenital cataracts to nephrogenic diabetes insipidus. More than 200 members of the aquaporin family have been found in plants, microbials, invertebrates and vertebrates, and their importance to the physiology of these organisms is being uncovered.

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Aquaporin water channels

Kozono David, Yasui Masato, King Landon S., Agre Peter

Journal of Clinical Investigation 109 （ 11 ） 1395 - 1399 2002年

ISSN 0021-9738

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Chapter 1 discovery of the aquaporins and their impact on basic and clinical physiology

Agre Peter, Borgnia Mario J., Yasui Masato, Neely John D., Carbrey Jennifer, Kozono David, Beitz Eric, Hoffert Jason, Leitch Virginia, King Landon S.

Current Topics in Membranes 51 1 - 38 2001年

ISSN 1063-5823

Functional impairment of lens aquaporin in two families with dominantly inherited cataracts

Francis Peter, Chung Jean Ju, Yasui Masato, Berry Vanita, Moore Anthony, Wyatt M. Keith, Wistow Graeme, Bhattacharya Shomi S., Agre Peter

Human Molecular Genetics 9 （ 15 ） 2329 - 2334 2000年09月

ISSN 0964-6906

Opacities in the crystalline lens of eye appear with high frequency in the general population. Dominantly inherited cataracts with differing clinical features were found in two families carrying different point mutations in the gene encoding lens water channel protein AQP0 (major intrinsic protein, MIP). Families with E134G have a uni-lamellar cataract which is stable after birth, whereas families with T138R have multi-focal opacities which increase throughout life. To establish pathophysiological relevance of cataract formation, the Xenopus laevis oocyte expression system was employed to evaluate functional defects in the mutant proteins, E134G and T138R. Both substitutions cause loss of membrane water channel activity due to impaired trafficking of the mutant proteins to the oocyte plasma membrane. Although missense mutations in AQP1 and AQP2 proteins are known to result in recessive traits in vivo and in vitro, when E134G or T138R are co-expressed with wild-type AQP0 protein, the mutant proteins exhibit dominant negative behaviour. To our knowledge, these studies represent the first in vitro demonstration of functionally defective AQP0 protein from humans with congenital cataracts. Moreover, these observations predict that less severe defects in the AQP0 protein may contribute to lens opacity in patients with common, less fulminant forms of cataracts.

Aquaporins in health and disease

King Landon S., Yasui Masato, Agre Peter

Molecular Medicine Today 6 （ 2 ） 60 - 65 2000年

ISSN 1357-4310

The molecular basis of membrane water-permeability remained elusive until the recent discovery of the aquaporin water-channel proteins. The fundamental importance of these proteins is suggested by their conservation from bacteria through plants to mammals. Ten mammalian aquaporins have thus far been identified, each with a distinct distribution. In the kidney, lung, eye and brain, multiple water-channel homologs are expressed, providing a network for water transport in those locations. It is increasingly clear that alterations in aquaporin expression or function can be rate-limiting for water transport across certain membranes. Aquaporins are likely to prove central to the pathophysiology of a variety of clinical conditions from diabetes insipidus to various forms of edema and, ultimately, they could be a target for therapy in diseases of altered water homeostasis.

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Regulation of AQP6 mRNA and protein expression in rats in response to altered acid-base or water balance

Promeneur Dominique, Kwon Tae Hwan, Yasui Masato, Kim Gheun Ho, Frøkiæer Jørgen, Knepper Mark A., Agre Peter, Nielsen Søren

American Journal of Physiology - Renal Physiology 279 （ 6 48-6 ） 2000年

ISSN 0363-6127

In the rat, aquaporin-6 (AQP6) is mainly localized in intercalated cells (ICs) in collecting ducts, where it is exclusively associated with intracellular vesicles. In this study, we examined whether AQP6 protein and mRNA expression were regulated in the inner medulla or inner stripe of the outer medulla. Rats treated with dietary alkali or acid load for 7 days with a fixed daily water intake revealed appropriate changes in urine pH but unchanged urine output. AQP6 protein and mRNA abundance were increased in alkali-loaded rats (187 ± 18 and 151 ± 17% of control, respectively), whereas no changes were observed in acid-loaded rats. Immunohistochemistry revealed increased IC AQP6 labeling in alkali-loaded rats but not in acid-loaded rats. In contrast, administration of NH₄Cl in the drinking water for 2 wk (free access to water) revealed a significant increase in AQP6 protein abundance (194 ± 9% of control), but this was associated with increased water intake. Combined, this suggests that AQP6 expression was not affected by acid loading per se but rather was in response to changes in water intake. Consistent with this, water loading for 48 h was associated with increased AQP6 protein abundance, compared with thirsted rats. Moreover, rats with lithium-induced nephrogenic diabetes insipidus had a threefold increase in both AQP6 protein and mRNA expression. Overall, these results suggest that AQP6 expression in collecting duct ICs is regulated by altered acid/alkali load or water balance. Thus AQP6 may contribute to maintenance of acid-base homeostasis and water balance.

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安井 正人

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論文 - 安井　正人