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, ␤-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking ␣-syntrophin (␣-Syn ؊/؊ ). The total level of AQP4 expression appears normal in brains of ␣-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 ␣-Syn ؊/؊ mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in ␣-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 ␣-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.

The water channel AQP4 is concentrated in perivascular and subpial membrane domains of brain astrocytes. These membranes form the interface between the neuropil and extracerebral liquid spaces. AQP4 is anchored at these membranes by its carboxyl terminus to ␣-syntrophin, an adapter protein associated with dystrophin. To test functions of the perivascular AQP4 pool, we studied mice homozygous for targeted disruption of the gene encoding ␣-syntrophin (␣-Syn ؊/؊ ). These animals show a marked loss of AQP4 from perivascular and subpial membranes but no decrease in other membrane domains, as judged by quantitative immunogold electron microscopy. In the basal state, perivascular and subpial astroglial end-feet were swollen in brains of ␣-Syn ؊/؊ mice compared to WT mice, suggesting reduced clearance of water generated by brain metabolism. When stressed by transient cerebral ischemia, brain edema was attenuated in ␣-Syn ؊/؊ mice, indicative of reduced water influx. Surprisingly, AQP4 was strongly reduced but ␣-syntrophin was retained in perivascular astroglial end-feet in WT mice examined 23 h after transient cerebral ischemia. Thus ␣-syntrophin-dependent anchoring of AQP4 is sensitive to ischemia, and loss of AQP4 from this site may retard the dissipation of postischemic brain edema. These studies identify a specific, syntrophin-dependent AQP4 pool that is expressed at distinct membrane domains and which mediates bidirectional transport of water across the brain-blood interface. The anchoring of AQP4 to ␣-syntrophin may be a target for treatment of brain edema, but therapeutic manipulations of AQP4 must consider the bidirectional water flux through this molecule. C erebral edema is essentially a loss of water homeostasis entailing a net increase of water flux into the brain. The route of water influx in this life-threatening condition is unknown, and no efficient therapy exists. We have previously shown that the brain expresses a water channel molecule, AQP4, that is strongly enriched in those astrocyte membrane domains forming the interface between brain neuropil and extracerebral spaces filled with blood or cerebrospinal fluid (1-3). To determine whether the pools of AQP4 in these specialized membrane domains are responsible for the fast influx of water that occurs during the development of brain edema, one must specifically eliminate the perivascular and subpial pools of AQP4 while leaving other pools of AQP4 intact. This can be achieved by deletion of ␣-syntrophin (␣-syn), an adapter protein in the dystrophin-associated protein complex that is required for anchoring AQP4 at these specialized membrane domains (4). Mice homozygous for targeted disruption of the gene encoding ␣-syntrophin (␣-Syn Ϫ/Ϫ ) exhibit a marked reduction of AQP4 in perivascular and subpial membranes but not in other locations in brain, because total brain AQP4 protein content is not reduced (4).The first aim of the present study was to use ␣-Syn Ϫ/Ϫ mice to investigate whether a selective depletion of the perivascular AQP4 pool reduces the vol...