Dynamics of membrane channels as revealed by freeze-fracture replica labeling

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Glutamate is known to cause swelling of astrocytes both in situ and in vitro. Although several different types of ionotropic and metabotropic glutamate receptors are expressed on astrocytes, their functional role in the glutamate-induced cell swelling is poorly understood. Using freeze-fracture replica immunoelectron microscopy, we have found a distinctive co-localization of metabotropic glutamate receptor 3 (mGluR3) and water channel aquaporn 4 (AQP4) on a specialized membrane domain, called orthogonal arrays of particles (OAPs), of astrocyte membranes in situ. To examine whether mGluR3 and AQP4 assemble to form OAPs, their cDNAs were introduced into Chinese hamster ovary cells. Replica immunoelectron microscopy revealed that both molecules were highly concentrated in OAPs-like structure. Agonists of mGluR3, glutamate and DCG-IV, disrupted co-localization of mGluR3 and AQP4, and decreased the density of OAPs-like structure in these cells. Similar effects of the agonists were also observed in astrocytes in situ. Functional analysis using the transfected cells showed that the agonists increased water permeability across cell membranes leading to cell swelling, probably in a cAMP-dependent manner. These findings suggest that mGluR3 and AQP4 assemble to form OAPs, and changes in the structural and functional states are governed by the activity of mGluR3. Therefore, we propose a pathway, mGluR3-AQP4 coupling, of osmotic cell volume regulation, of astrocytes.