Expression of multiple AQP4 pools in the plasma membrane and their association with the dystrophin complex

Abstract

Altered aquaporin-4 (AQP4) expression has been reported in brain edema, tumors, muscular dystrophy, and neuromyelitis optica. However, the plasma membrane organization of AQP4 and its interaction with proteins such as the dystrophin-associated protein complex are not well understood. In this study, we used sucrose density gradient ultracentrifugation and 2D blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the expression of several AQP4 multi-subunit complexes (pools) of different sizes, ranging from >> 1 MDa to similar to 500 kDa and containing different ratios of the 30/32 kDa AQP4 isoforms, indicative of orthogonal arrays of particles of various sizes. A high molecular weight pool co-purified with dystrophin and beta-dystroglycan and was drastically reduced in the skeletal muscle of mdx3cv mice, which have no dystrophin. The number and size of the AQP4 pools were the same in the kidney where dystrophin is not expressed, suggesting the presence of dystrophin-like proteins for their expression. We found that AQP2 is expressed only in one major pool of similar to 500 kDa, indicating that the presence of different pools is a peculiarity of AQP4 rather than a widespread feature in the AQP family. Finally, in skeletal muscle caveolin-3 did not co-purify with any AQP4 pool, indicating the absence of interaction of the two proteins and confirming that caveolae and orthogonal arrays of particles are two independent plasma membrane microdomains. These results contribute to a better understanding of AQP4 membrane organization and raise the possibility that abnormal expression of specific AQP4 pools may be found in pathological states

Aquaporin-4 expression is severely reduced in human sarcoglycanopathies and dysferlinopathies

“…This suggest that larger OAPs, that are mainly composed of the M23 isoform are preferentially affected in the dystrophic muscle. 32 AQP4 was previously shown to be decreased in dystrophindeficient muscle tissues, nevertheless, our group of cases displayed normal or just slightly reduced dystrophin levels, thus indicating that the disruption of the sarcoglycan complex per se can induce a change in AQP4 expression.…”

Evidences for a Leaky Scanning Mechanism for the Synthesis of the Shorter M23 Protein Isoform of Aquaporin-4

“…BN/SDS-PAGE-Polyacrylamide native gradient gels (3-9%) were prepared as described (40,41). Twenty g of protein sample prepared as described earlier were mixed with 5% of Coomassie Blue G-250 and loaded in each lane.…”

“…When coexpressed, they form OAPs of intermediate size, similar to those seen in native tissues, suggesting that the ratio of the two isoforms is central to the determination of OAP size (14,37). Biochemical analysis using blue native (BN)/SDS-PAGE has recently shown the expression of several AQP4 multisubunit complexes (pools) ranging from several MDa to ϳ400 kDa in the brain, indicative of OAPs of various sizes (40,41). These pools contain different ratios of the 30/32-kDa isoforms.…”

Identification of a Point Mutation Impairing the Binding between Aquaporin-4 and Neuromyelitis Optica Autoantibodies

“…Immunoprecipitation-Rat brain (cerebral cortex) membrane vesicles and skeletal muscle light microsome vesicles were prepared as reported (39). Both AQP4-transfected cells and plasma membrane vesicles were extracted using IP buffer (500 mM 6-aminohexanoic acid, 50 mM imidazole pH 7, 2 mM EDTA, 150 mM NaCl, 3% n-dodecyl ␤-D-maltoside), and added with protease inhibitor mixture (Roche Diagnostic Gmbh, Mannheim, Germany).…”