Published papers

Discovery of aquaporin water channel proteins has provided insight into the molecular mechanism of membrane water permeability. In mammalian brain, Aquaporin-4 (AQP4) is the main water channel and is distributed with highest density in the perivascular and subpial astrocyte end-feet. AQP4 is a critical component of an integrated water and potassium homeostasis. Indeed, AQP4 has been implicated in several neurologic conditions, such as brain edema, seizure and even mood disorders. Expression and regulation of AQP4 have been studied to understand the roles of AQP4 in physiological and pathological conditions. Here we discuss about the mechanisms how AQP4 is dynamically regulated at different levels; channel gating, subcellular distribution, phosphorylation, protein-protein interactions and orthogonal array formation. Interestingly, AQP4 has been identified as a target antigen of autoimmune attack in neuromyelitis optica (NMO). We have evaluated putative epitopes on AQP4 for NMO-IgG binding. We have also studied Drosophila Big Brain (Bib), since Bib has high sequence homology to AQP-4, and play an important role for Drosophila neurogenesis. AQP4 may be a potential therapeutic target in several neurologic conditions. Further studies from different aspects are required to develop new drugs against AQP4.

Aquaporin-4 (AQP4) is the predominant water channel in the brain. Although AQP4 plays an important role in brain water homeostasis, the molecular mechanisms of AQP4 regulation are not fully understood. In this report, we show how Zn2+ rapidly and reversibly decreases the water permeability of AQP4 when it is reconstituted into proteoliposomes. Mutagenesis analysis identified Cys178, located in cytoplasmic loop D, as a target residue of ZnCl2 inhibition. Moreover, treatment with diamide enhanced the inhibitory effects of ZnCl2. These results suggest that the water permeability of AQP4 may be regulated by dynamic changes in intracellular Zn2+ concentration linked to the cellular redox state. © 2009 American Chemical Society.

The amniotic membrane encloses and retains amniotic fluid during pregnancy. In general, fluid flux is regulated by epithelial tissues, which have tight junctions (TJs). However, TJs have not yet been identified in the amniotic epithelium. In this study, we have determined whether the mouse amniotic epithelium contains TJs. Freeze-fracture electron microscopy revealed the presence of strand-like TJs in the amniotic epithelium. Amniotic TJs were composed of occludin; zona occludens (ZO)-1; and claudins 1, 3, 4, and 7. These claudins underwent developmental changes during pregnancy. The localization patterns of the claudins and their detergent solubility drastically changed between embryonic day (E) 16 and E17; the volume of the amniotic fluid also decreased sharply. Furthermore, in vitro assessment of amniotic membrane permeability showed that the amniotic membrane was more permeable on E17 than on E16. On E17, TJ components were sparsely distributed in parts of the amniotic epithelium. The results of Annexin V-fluorescein staining and Terminal dUTP nick-end labeling (TUNEL) assay revealed ongoing apoptosis in all the cells in such regions. The above findings suggest that TJs in the amniotic epithelium maintain amniotic fluid volume during pregnancy, while apoptosis of amniotic epithelial cells between E16 and E17 causes disruption of the TJs. © 2009 Elsevier Ltd. All rights reserved.

Homeostasis of neuronal activity is crucial to neuronal physiology. In dendrites, hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 1 is considered to play critical roles in this process. While electrophysiological studies have demonstrated the dynamic modulation of Ih current mediated by HCN1 proteins, little is known about the underlying molecular and cellular mechanisms. In this study, we utilized cortical cultured neurons and biochemical methods to identify molecular and cellular mechanisms that mediate the physiological regulation of HCN1 channel functions in cortical neurons. Pharmacological manipulations of neuronal activity resulted in changes in the expression level of HCN1. In addition, the surface expression of HCN1 was dynamically regulated by neuronal activity. Both of these changes led to functional modulations of HCN1 channels. Our study suggests that coordinated changes in protein expression and surface expression of HCN1 serve as the key regulatory mechanisms controlling the function of endogenous HCN1 protein in cortical neurons. © 2009 Elsevier Inc. All rights reserved.

The neurogenic gene Drosophila big brain (bib) has a high sequence homology to aquaporin-4. However, its cellular functions in Drosophila neurogenesis have remained elusive. Here we investigated cell adhesion, and the ion and water permeability of Bib. The adhesive function was examined by a cell aggregation assay using L cells. Bib-transfected L cells formed aggregated clusters, while control-L cells remained as a single cell suspension. Ion permeation was not confirmed in L cells stably expressing Bib. When expressed in COS7 cells, Bib exhibited limited water permeability. This newly found cell adhesive function of Bib may be important for Drosophila neurogenesis. © 2009 Federation of European Biochemical Societies.

Aquaporin 6 (AQP6) is an anion channel that is expressed primarily in acid secreting α-intercalated cells of the kidney collecting duct. In addition, AQP6 anion channel permeability is gated by low pH. Inspection of the N-terminus of AQP6 revealed a putative calmodulin binding site. AQP6-expressing CHO-K1 cell lysates were mixed with calmodulin beads and AQP6 was pulled down in the presence of calcium. Mutagenesis of the N-terminal calmodulin binding site in full length mouse AQP6 resulted in a loss of calmodulin binding activity. Mouse and human AQP6 calmodulin binding site peptides bound dansyl-calmodulin with a dissociation constant of approximately 1 μM. The binding of AQP6 to calmodulin may be an important key to determining the physiological role of AQP6 in the kidney. © 2009 Elsevier Inc. All rights reserved.

Context: The prevalence of congenital hypothyroidism (CH) associated with mutations in the TSH receptor gene (TSHR) has not been established. Objective: We examined the frequency of TSHR mutations among patients with permanent primary CH and in the general population in Japan. Subjects and Methods: We enrolled 102 patients with permanent primary CH [70 with "moderate to severe CH" (TSH, >10 mU/liter) and 32 with "mild CH" (TSH, 5-10 mU/liter)], who were identified through newborn screening among 353,000 newborns born in Kanagawa prefecture from October 1979 to June 2006. These subjects were tested for TSHR mutations by PCR-based direct sequencing. We further characterized molecular functions of identified mutant TSHRs in vitro. Results: We found three patients with moderate to severe CH who had biallelic mutations in TSHR and three patients with mild CH who had monoallelic mutations. Observed mutations included one previously characterized mutation (p.R450H) and three uncharacterized mutations (p.G132R, p.A204V, and p.D403N). In vitro experiments confirmed loss of functions of these four mutants. Among four mutations, p.R450H was particularly frequent: six of nine mutant alleles harbored p.R450H. All six alleles with p.R450H commonly carried a minor single nucleotide polymorphism, suggesting a founder effect. We estimated the prevalence of biallelic TSHR mutations to be 4.3% (three in 70) in Japanese patients with moderate to severe CH, and 1 in 118,000 (three in 353,000) in the general Japanese population. Conclusions: In Japan, TSHR mutations are relatively common among patients with CH, and a founder mutation (p.R450H) accounts for about 70% of mutants. Copyright © 2009 by The Endocrine Society.

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.

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 (Pf) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl2-. 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 Pf 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 Pf 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.

How do lipid molecules in membranes perform a flip-flop? The flip-flops of lipid molecules play a crucial role in the formation and flexibility of membranes. However, little has been determined about the behavior of flip-flops, either experimentally, or in molecular dynamics simulations. Here, we provide numerical results of the flip-flops of model lipid molecules in a model membrane and investigate the statistical properties, using millisecond-order coarse-grained molecular simulations (dissipative particle dynamics). We find that there are three different ways of flip-flops, which can be clearly characterized by their paths on the free energy surface. Furthermore, we found that the probability of the number of the flip-flops is well fitted by the Poisson distribution, and the probability density function for the inter-occurrence times of flip-flops coincides with that of the forward recurrence times. These results indicate that the occurrence of flip-flops is a Poisson process, which will play an important role in the flexibilities of membranes. © 2014 AIP Publishing LLC.

Interionic distances are shorter in concentrated ionic solutions, thus instigating the interaction and overlap of hydration shells, as ions become separated by only one or two layers of water molecules. The simultaneous interaction of water with two oppositely charged ions has, so far, only been investigated by computer simulation studies, because the isolated vibrational spectroscopic signature of these molecules remains undetected. Our combined near-infrared spectroscopic and molecular dynamics simulation studies of alkali halide solutions present a distinct spectral feature, which is highly responsive to depletion of bulk water and merging of hydration shells. The analysis of this spectral feature demonstrates that absorption trends are in good agreement with the law of matching affinities, thus providing the first successful vibrational spectroscopic treatment of this topic. Combined with commonly observed near-infrared bands, this feature provides a spectral pattern that describes some relevant aspects of ionic hydration.

Background and Aim: Basolateral water channel, aquaporin-4 (AQP4), is known to be expressed in gastric parietal cells, especially in the basal side of gastric mucosa. However, the role of AQP4 in the stomach is still unknown. Histamine type 2 receptor (H2R) knockout mice, which are characterized by suppressed gastric acid secretion, are known as formation of mucosal hyperplasia with cystic dilatation and spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach. The aim of the present study is to investigate whether the expression of AQP4 is changed by the condition of acid suppression and Helicobacter pylori infection. Methods: Male H2R knockout mice and their controls (C57BL/6) were used. H.pylori was orally infected at the age of 5 weeks. The distributions of AQP4 and H+/K+-ATPase in the gastric mucosa were investigated by fluorescent immunohistochemistry. The mRNA expressions of AQP4, H+/K+- ATPase, sonic hedgehog (Shh), and trefoil factor-2 (TFF2) were investigated by quantitative reverse transcription polymerase chain reaction (RT-PCR). Results: In the H2R knockout mice, the distribution of AQP4-positive parietal cells was extended toward the surface of the fundic glands. Although the mRNA expression levels of AQP4 and H+/K+ ATPase were elevated in H2R knockout mice at the age of 20 weeks, the elevations were not maintained by aging or H.pylori infection. In H2R knockout mice with H.pylori infection, the expression level of TFF2mRNA was elevated while the ratio between AQP4 and H+/K+ ATPase mRNA expression was decreased compared with the H2R knockout mice without H.pylori infection. Conclusions: In the H2R knockout mice, massive SPEM was induced by H.pylori colonization and the ratio between AQP4 and H+/K+ ATPase mRNA expression was decreased.

Epilepsy is characterized by the abnormal activation of neurons in the cerebral cortex, but the molecular and cellular mechanisms contributing to the development of recurrent seizures are largely unknown. Recently, the critical involvement of astrocytes in the pathophysiology of epilepsy has been proposed. However, the nature of plastic modulations of astrocytic proteins in the epileptic cortex remains poorly understood. In this study, we utilized the zero magnesium in vitro model of epilepsy and examined the potential molecular changes of cortical astrocytes, focusing specifically on endfeet, where specialized biochemical compartments exist. We find that the continuous epileptic activation of neurons for 1 h decreases the expression level of β-dystroglycan (βDG) in acute cortical brain slices prepared from mice. This change is completely abolished by the pharmacological blockade of NMDA-type glutamate receptors as well as by matrix metalloproteinase inhibitors. Consistent with the highly specialized localization of βDG at astrocytic endfeet, where it plays a pivotal role in anchoring endfeet-enriched proteins in astrocytes, the down-regulation of βDG is accompanied by a decrease in the expression of AQP4 but not laminin. Importantly, this down-regulation of βDG persists for at least 1 h, even after the apparent recovery of neuronal activation. Finally, we show that the down-regulation of βDG is associated with the dysfunction of the endfeet at the blood-brain interface as a diffusion barrier. These results suggest that the sustained downregulation of βDG leads to dysfunctions of astrocytic endfeet in the epileptic cerebral cortex and may contribute to the pathogenesis of epilepsy.

Aquaporin-4 (AQP4) is a water channel protein that is predominantly expressed in astrocytes in the CNS. The rapid water flux through AQP4 may contribute to electrolyte/water homeostasis and may support neuronal activities in the CNS. On the other hand, little is known about the expression of AQP4 in the peripheral nervous system (PNS). Using AQP4-/- mice as a negative control, we demonstrated that AQP4 is also expressed in sensory ganglia, such as trigeminal ganglia and dorsal root ganglia in the PNS. Immunohistochemistry revealed that AQP4 is exclusively localized to satellite glial cells (SGCs) surrounding the cell bodies of the primary afferent sensory neurons in the sensory ganglia. Biochemical analyses revealed that the expression levels of AQP4 in sensory ganglia were considerably lower than those in astrocytes in the CNS. Consistently, behavioral analyses did not show any significant difference in terms of mechanical and cold sensitivity between wild type and AQP4-/- mice. Overall, although the pathophysiological relevance of AQP4 in somatosensory perception remains unclear, our findings provide new insight into the involvement of water homeostasis in the peripheral sensory system.

Water molecules play an important role in providing unique environments for biological reactions on cell membranes. It is widely believed that water molecules form bridges that connect lipid molecules and stabilize cell membranes. Using all-atom molecular dynamics simulations, we show that translational and rotational diffusion of water molecules on lipid membrane surfaces exhibit subdiffusion and aging. Moreover, we provide evidence that both divergent mean trapping time (continuous-time random walk) and long-correlated noise (fractional Brownian motion) contribute to this subdiffusion. These results suggest that subdiffusion on cell membranes causes the water retardation, an enhancement of cell membrane stability, and a higher reaction efficiency.

Aquaporins (AQPs), which transport water molecules across cell membranes, are involved in many physiological processes. Recently, it is reported that the water-water interactions within the channel are broken at the aromatic/arginine selectivity filter (ar/R region), which prevents proton transportation [U. K. Eriksson, Science 340, 1346 (2013)SCIEAS0036-807510.1126/science.1234306]. However, the effects of the conformational fluctuations of amino acids on water transportation remain unclear. Using all-atom molecular dynamics simulations, we analyze water transportation and fluctuations of amino acids within AQP1. The amino acids exhibit 1/f fluctuations, indicating possession of long-term memory. Moreover, we find that water molecules crossing the ar/R region obey a non-Poisson process. To investigate the effect of 1/f fluctuations on water transportation, we perform restrained molecular dynamics simulations of AQP1 and simple Langevin stochastic simulations. As a result, we confirm that 1/f fluctuations of amino acids contribute to water transportation in AQP1. These findings appreciably enhance our understanding of AQPs and suggest possibilities for developing biomimetic nanopores. © 2014 American Physical Society.

Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols. © 2014 Kato et al.