Abstract
The water-permeable channel aquaporin-4 (AQP4) is highly expressed in perivascular astrocytes of the mammalian brain and represents the major conduit for water across the blood-brain barrier. Within these cells, AQP4 is found in great quantities at perivascular endfoot sites but is detected in lesser amounts at the membrane domains within the brain parenchyma. We had previously established that this polarization was regulated by the interaction between dystroglycan (DG), an extracellular matrix receptor that is co-expressed with AQP4, and the laminin that is contained within the perivascular basal lamina. In the present study, we have attempted to describe the mechanisms that underlie this regulation, using primary astrocyte cultures. Via biotinylation, we found that the cell-surface expression of AQP4 is DG-dependent and is potentiated by laminin. We also determined that this laminin-dependent increase occurs not through an upregulation of total AQP4 levels, but rather from a redirection of AQP4 from an intracellular, EEA-1-associated pool to the cell surface. We then demonstrated an association between DG and dynamin and showed that dynamin functioned in conjunction with clathrin to regulate surface AQP4 amounts. Furthermore, we observed that DG preferentially binds to the inactive forms of dynamin, suggesting that this interaction was inhibitory for AQP4 endocytosis. Finally, we showed that laminin selectively upregulates the cell-surface expression of the M23 isoform of AQP4. Our data therefore indicate that the dual interation of DG with laminin and dynamin is involved in the regulation of AQP4 internalization, leading to its asymmetric enrichment at perivascular astrocyte endfeet.
Highlights
The aquaporins are a family of bidirectional water-permeable channels that are expressed in a wide variety of tissues
We saw that laminin selectively upregulates the M23, but not the M1 isoform of AQP4. Based on these lines of evidence, we propose that laminin, DG, and dynamin comprise the primary components of a complex involved in the regulation of clathrin-mediated AQP4 endocytosis, and that their interaction serves to promote the enrichment of M23-based orthogonal arrays of particles at astrocytic endfeet
We determined that primary astrocytes cultured in the presence of exogenous laminin, compared to untreated controls, did exhibit an approximately threefold (2.97 ± 0.37) increase in AQP4 amounts at the plasma membrane (Fig 1A left and B)
Summary
The aquaporins are a family of bidirectional water-permeable channels that are expressed in a wide variety of tissues. While animals that lack AQP4 expression exhibit no detectable behavioral defects or gross morphological disruptions in BBB structure [4], they do present deficits in olfaction and audition [5] [6], indicating that the channel may play a central role in brain function. Studies of these animals have revealed the importance of this channel in cases of severe insults to the brain, such as stroke or injury, in which cytotoxic and vasogenic edema can develop, and often are the main contributors to morbidity or death [7] [8]. The modulation of AQP4 activity or expression in the brain could result in the amelioration of these disease states
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