An α-syntrophin-dependent pool of AQP4 in astroglial end-feet confers bidirectional water flow between blood and brain

Abstract

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...

Potassium channel Kir4.1 macromolecular complex in retinal glial cells

“…For example, immunoelectron microscopy showed that the absence of a-syntrophin dramatically altered the localization pattern of AQP4 but not Kir4.1 in hippocampal astrocytes (Amiry-Moghaddam et al, 2003a). Similar evidence has been proposed in retinas from a-syntrophin null mice where the localization of AQP4 was marginally decreased in M€ uller cell endfeet and perivascular processes, whereas Kir4.1 localization was unaffected (Frydenlund et al, 2004).…”

“…Furthermore, AQP4 was shown to interact with the DGC in M€ uller cells (Dalloz et al, 2003) and brain astrocytes (Amiry-Moghaddam et al, 2003aNeely et al, 2001). Based on this evidence, experiments were conducted to examine whether AQP4 can interact with DGC proteins and also with Kir4.1 in retina.…”

“…Based on the previously shown interactions between AQP4 and a-syntrophin in brain (Amiry-Moghaddam et al, 2003a;Neely et al, 2001), AQP4 was first immunoprecipitated from rat retinal lysate, then probed with pan-syntrophin antibodies. The results of this experiment show that a protein slightly less than 75 kDa was recovered (Fig.…”

“…Recent findings suggest that AQP4 may also be necessary for effective [K 1 ] o buffering in brain (Amiry-Moghaddam et al, 2003a;Nagelhus et al, 2004). The co-localization of this channel with Kir4.1 in M€ uller cells suggests a common molecular mechanism for their consolidation in these subcellular regions (Nagelhus et al, 2004).…”

“…Studies by our group and others have revealed that both Kir4.1 (Connors and Kofuji, 2002;Connors et al, 2004) and AQP4 (Neely et al, 2001;Amiry-Moghaddam et al, 2003a) can interact with the multi-protein dystrophin-glycoprotein complex (DGC) in brain and astrocytes. This group of proteins has been best described in muscle, where it may be involved in calcium homeostasis or the maintenance of structural integrity required for myocytes to withstand muscular contractile forces (Mehler, 2000).…”