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WIREs Membr Transp Signal

Aquaporin‐4 orthogonal arrays of particles from a physiological and pathophysiological point of view

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Aquaporin‐4 (AQP4) is the neuromuscular water channel that is also expressed at the basolateral membranes of other cell types in kidney, stomach, and lung. In skeletal muscle, AQP4 is found at the sarcolemma of fast‐twitch fibers and its function is strictly correlated with the glycolytic metabolism. In the central nervous system, AQP4 is expressed at the basolateral membranes of ependymal cells, and is highly concentrated at the glial end‐foot processes surrounding blood vessels and forming the glia limitans, as well as at the nonend‐foot glial processes of the granule cell layer in the cerebellum. AQP4 plasma membrane organization is different from other aquaporins (AQPs). AQP4 is expressed as two major polypeptides called M1 and M23. These two isoforms form heterotetramers appearing in the plasma membrane as intramembrane particles (IMPs) observable by freeze‐fracture electron microscopy. Such tetrameric organization is common to all other AQPs. In the case of AQP4, however, multiple IMPs further aggregate to form structures called orthogonal arrays of particles (OAPs). The relative abundance of M23 and M1 in vivo is the major determinant for the formation of OAPs of different sizes. The function of AQP4 aggregation into OAPs under normal conditions is still not completely understood. Interestingly, there are several reports indicating that OAPs are involved in different neuromuscular diseases. In particular, the OAP‐related diseases that have attracted more attention are Duchenne muscular distrophy and, more recently, neuromyelitis optica, the two pathological conditions in which OAPs are involved in completely different ways. WIREs Membr Transp Signal 2013, 2:143–154. doi: 10.1002/wmts.86

Conflict of interest: The authors have declared no conflicts of interest for this article.

From Aquaporins (AQP) tetrameric organization to AQP4 orthogonal arrays of particles (OAPs). (a) AQP1 exists as a 28 kDa glycosylated (gly) isoform whereas AQP4 (b) exists as two major isoforms, called M23 and M1, of 30 and 32 kDa respectively. AQP4 is not glycosylated. (c) AQP1, like all AQPs, is organized in tetramers in which each monomer is a functional unit for water transport and forms homotetramers, made of the same isoform. By freeze‐fracture electron microscopy (FFEM), AQPs appear as intra‐membrane particles (IMPs), as shown for AQP1 in the image on the right. (d) AQP4 is unique as it forms heterotetramers made of the two major isoforms of 30 and 32 kDa. As shown in the image on the right, AQP4‐IMPs are able to further aggregate into higher order structures called orthogonal arrays of particles (OAPs) or square arrays (white circles). (e) OAPs of different sizes can be biochemically separated by BN/SDS‐PAGE. Using a 4–9% gradient gel in the first dimension, performed under native conditions, seven different OAP pools can be visualized. The densitometric analysis of M1 and M23 bands, performed for each of the seven pools, shows that the smaller the size of the OAP pool, the higher is the M1/M23 ratio. (The FFEM images: Reprinted with permission from Ref . Copyright 2012 Springer for AQP1 in (c) and Reprinted with permission from Ref . Copyright 2012 National Academy of Science for AQP4. The images in (e): Reprinted with permission from Ref . Copyright 2012 John Wiley and Sons)
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Neuromyelitis optica (NMO)–IgG does not block aquaporin‐4 (AQP4) water transport. (a) Primary cultured astrocytes were incubated with Multiple Sclerosis (MS) and NeuroMyelitis Optica (MNO) sera for 15 min, 1 and 2 days at a dilution of 1:500 in culture medium. No significant differences were found between the conditions. The cell swelling was significantly slowed only in AQP4 siRNA‐treated astrocytes (astrocytes AQP4 KD) on day 6 of RNA interference (the histograms show the mean +/− SE (standard error) of separate sets of measurements, n = 3–7, *P < 0.01). (b) TNC‐1 cells transfected with M23‐AQP4 isoform were treated as described in (a) for astrocytes. Typical swelling kinetics (left) and histogram (right) of the time constant (s) of the exponential curve fitting after hypotonic shock. No significant differences in cell swelling kinetics were found between the different conditions. (Reprinted with permission from Ref . Copyright 2012 John Wiley and Sons)
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Neuromyelitis optica (NMO–IgG epitope structures. Drawings of an aquaporin‐4 orthogonal arrays of particle (AQP4‐OAP) (a) and AQP4 tetramer (b). When AQP4 organizes into OAPs the extracellular loops of each tetramer interact and create at least two different NMO–IgG epitopes.
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Indication that orthogonal arrays of particles (OAPs) are the target for neuromyelitis optica (NMO) autoantibodies. (a) NMO labeling in spinal cord and brain sections is much stronger at the perivascular astrocytes, characterized by the largest OAPs (arrows) more visible in the white matter (wm) and in the molecular cell layer (mcl). In contrast, the staining is very weak at the astrocyte process network (arrowheads) in the granular cell layer (gcl) and gray matter (gm). (b) Experiments performed on astrocyte primary cultures and HeLa cells stably transfected with the M1 or M23‐AQP4 isoform showing that NMO–IgGs selectively recognize OAP expressing cells (b). (c) BN/SDS‐PAGE and immunofluorescence experiments showing that NMO–IgGs selectively recognize AQP4‐OAPs in rat stomach whereas they do not recognize AQP4‐tetramers in mouse stomach. (d) Immunofluorescence experiments performed on fluorescent AQP4 fusion proteins, mCherry C‐terminus‐tagged AQP4 (able to form OAPs) and green fluorescent protein (GFP)‐N‐terminus tagged AQP4 (unable to form OAPs). NMO–IgGs only recognize mCherry‐AQP4. (The images in panels (a)–(c): Reprinted with permission from Ref . Copyright 2012 John Wiley and Sons. The images in panel (d): Reprinted with permission from Ref . Copyright 2012 The American Society for Biochemistry and Molecular Biology)
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Aquaporin‐4 (AQP4) reduction and astrocyte swelling in mdx mice. Immunogold experiments performed in the brain of mdx mice (b) and relative control (a) with anti‐AQP4 antibodies visualized by gold particles indicated by the arrows. Note that the number of gold particles is strongly reduced in mdx perivascular astrocytes. Moreover, this reduction is associated with swollen perivascular astrocyte (asterisks). (Reprinted with permission from Ref . Copyright 2012 The Faseb Journal)
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Possible aquaporin‐4 (AQP4) interacting proteins: dystrophin associated proteins (DAPs) and actin cytoskeleton. (a) BN/SDS‐PAGE followed by immunoblot analysis showing that the glial dystrophin isoform (DP71), β‐dystroglycan (βDG), and AQP4 colocalize in the largest pool of orthogonal arrays of particles (OAPs) (arrow). (b) The possible interaction between AQP4 and actin cytoskeleton in cultured astrocytes seems to depend on the cell adhesion phase and is therefore correlated with rapid versus slow changes in cell morphology. Immediately after plating (round cells), F‐actin and AQP4 colocalize in Control (CTRL) conditions and cytochalasin‐D treatment completely impairs AQP4 plasma membrane localization. In contrast, after 1 day, when astrocytes become flat with a fibroblast‐like shape, cytochalasin‐D treatment does not seem to affect AQP4 localization. (The BN‐PAGE analysis: Reprinted with permission from Ref . Copyright 2012 John Wiley and Sons). (The images in (b): Reprinted with permission from Ref . Copyright 2012 John Wiley and Sons)
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The importance of aquaporin‐4 orthogonal arrays of particles (AQP4‐OAPs) in cell physiology. (a) Schematic drawing showing that AQP4 aggregation into OAPs could serve to enhance the water permeability per surface unit. (b) FRAP experiment performed on cells transfected with the M1 isoform, organized into AQP4 tetramers (c), or M23 isoform able to aggregate into very large OAPs (d). The recovery of fluorescence in the bleached area takes 5 min for M1 tetramers (c), whereas almost no recovery is shown for ‘immobile’ M23 (d). (e) Lateral diffusion data are shown as reciprocal half‐times (t1/2) for fluorescence recovery (mean +/− SE of separate sets of measurements, n = 3–7. The schematic drawing in (d) shows that large OAPs may form macro‐molecular complexes, with the dystrophin associated protein complex. (The FRAP images (b) and the fluorescence recovery analysis (c–e): Reprinted with permission from Ref . Copyright 2012 The American Society for Biochemistry and Molecular Biology)
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