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. 2001 Nov 20;98(24):14108-13.
doi: 10.1073/pnas.241508198.

Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein

J D Neely  1 , M Amiry-Moghaddam, O P Ottersen, S C Froehner, P Agre, M E Adams
Affiliations

Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein

J D Neely et al. Proc Natl Acad Sci U S A. .

Abstract

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, beta-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking alpha-syntrophin (alpha-Syn(-/-)). The total level of AQP4 expression appears normal in brains of alpha-Syn(-/-) mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of alpha-Syn(-/-) mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in alpha-Syn(-/-) mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse-chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires alpha-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.

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Figures

Figure 1
Figure 1
Immunoblots of brain and skeletal muscle. (A) Coimmunoprecipitation (IP) of AQP4 with syntrophin and other dystrophin-associated proteins from dithiobis(succinimidyl propionate)-stabilized rat cerebellum homogenate by AQP4 or pan-syntrophin antibodies. Immunoblots (IB) showed specific reactions with antibodies to AQP4 (major band, 32 kDa), β-dystroglycan (βDG, 43 kDa), or dystrophin isoform (Dp71, 71 kDa). Nonspecific IgG immunoprecipitates were nonreactive. (B) AQP4 immunoblots of cerebellum and cerebral cortex membranes (20 μg protein per lane) and AQP4 immunoprecipated from skeletal muscle of wild-type (wt) mice or mice lacking α-Syn (Syn−/−).
Figure 2
Figure 2
Immunogold electron micrographs of cerebellum from heterozygous (α-Syn+/−) and α-Syn-null (α-Syn−/−) mice incubated with antibodies to AQP4 (AQP4 Ab) or α-Syn (Syn Ab). (A and B) AQP4 immunolabeling of astrocyte perivascular endfeet in membranes facing endothelial basal lamina (paired arrowheads) or membranes facing neuropil (double arrowheads). (C and D) α-Syn immunolabeling is similar to AQP4 in wild-type (wt) mice but is absent in α-Syn−/− mice. End, capillary endothelial cell; N, nucleus; L, lumen. (Scale bars, 0.5 μm.)
Figure 3
Figure 3
Anti-AQP4 immunogold electron micrographs of cerebral cortex from (A) wild-type (wt) and (B) α-Syn-null (α-Syn−/−) mice. Anti-AQP4 labeling of astrocyte endfeet in membranes facing endothelial basal lamina (paired arrowheads) or membranes facing neuropil (double arrowheads). End, capillary endothelial cell; N, nucleus; L, lumen. (Scale bars, 0.5 μm.)
Figure 4
Figure 4
Confocal anti-AQP4 immunofluorescence of skeletal muscle from wild-type (wt) and α-Syn-null (α-Syn−/−) mice. Quadriceps femoris labeled with AQP4 and α-Syn antibodies reveals nearly complete loss of AQP4 from fast-twitch fibers in the absence of α-Syn.
Figure 5
Figure 5
AQP4 expression in cell lines transfected with wild-type (wt) AQP4 or AQP4 lacking three C-terminal residues (ΔSSV). (A) Transfected HEK293, CHO-K1, and MDCK cells labeled with anti-AQP4 and analyzed by immunofluorescence confocal microscopy show AQP4 wt and ΔSSV localized to the plasma membrane. Basolateral targeting in MDCK cells was confirmed in vertical sections (see Insets). (B) AQP4 immunoblot of membrane proteins prepared in duplicate from transfected HEK293 cells (20 μg protein per lane). (C) Expression of AQP4, neomycin resistance gene (NeoR), and β-tubulin (β-tub) assessed by RT-PCR of RNA from transfected HEK293 cells and ethidium bromide staining of agarose gels.
Figure 6
Figure 6
Pulse–chase metabolic labeling of stably transfected HEK293 cells expressing wild-type (wt) AQP4 or AQP4 lacking three C-terminal residues (ΔSSV). (A) Autoradiograph of cell lysates analyzed by AQP4 immunoprecipitation and SDS/PAGE. (B) Graph of the amount of [35S]methionine-labeled AQP4 protein in the cells during chase. Two independent experiments (open and filled symbols) are shown. wt AQP4 (○); AQP4 ΔSSV (▿).
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