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

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, ␤-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.

Potassium channel Kir4.1 macromolecular complex in retinal glial cells

“…In addition, immunolocalization studies have shown that Kir4.1 and AQP4 are tightly co-localized in rat retina (Nagelhus et al, 1999), and AQP4 localization in astrocytes appears to be highly dependent on a-syntroin (Inoue et al, 2002). Direct interactions between AQP4, a-syntrophin, Dp71 and b-dystroglycan have been shown by IP assays (Neely et al, 2001;Amiry-Moghaddam et al, 2003a,b). Finally, the specific loss of Dp71 eliminates the polarized distribution of both Kir4.1 and AQP4 in retina (Dalloz et al, 2003).…”

“…We employed the crosslinking reagent DSP for the current studies to minimize instability of interactions between channel and the DGC. This reagent has previously been used by other groups to stabilize interactions between the transmembrane channels and their interaction partners (Zhu et al, 1999;Neely et al, 2001). Whole retinas from rats were isolated, chemically crosslinked, then incubated with antibodies against Kir4.1.…”

“…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.…”

“…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).…”