Abstract
Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the central nervous system (CNS). It is predominantly expressed in astrocytes lining blood–brain and blood–liquor boundaries. AQP4a (M1), AQP4c (M23), and AQP4e, present in the plasma membrane, participate in the cell volume regulation of astrocytes. The function of their splicing variants, AQP4b and AQP4d, predicted to be present in the cytoplasm, is unknown. We examined the cellular distribution of AQP4b and AQP4d in primary rat astrocytes and their role in cell volume regulation. The AQP4b and AQP4d isoforms exhibited extensive cytoplasmic localization in early and late endosomes/lysosomes and in the Golgi apparatus. Neither isoform localized to orthogonal arrays of particles (OAPs) in the plasma membrane. The overexpression of AQP4b and AQP4d isoforms in isoosmotic conditions reduced the density of OAPs; in hypoosmotic conditions, they remained absent from OAPs. In hypoosmotic conditions, the AQP4d isoform was significantly redistributed to early endosomes, which correlated with the increased trafficking of AQP4-laden vesicles. The overexpression of AQP4d facilitated the kinetics of cell swelling, without affecting the regulatory volume decrease. Therefore, although they reside in the cytoplasm, AQP4b and AQP4d isoforms may play an indirect role in astrocyte volume changes.
Highlights
Aquaporin-4 (AQP4) is a transmembrane channel that selectively transports water molecules and is expressed in different cell types throughout the body [1,2,3,4,5,6]
We expressed recombinant AQP4b and AQP4d isoforms encoded by fusion proteins AQP4b-GFP
We show that isoforms AQP4b and AQP4d reside in intracellular structures and remain there in hypoosmotic conditions
Summary
Aquaporin-4 (AQP4) is a transmembrane channel that selectively transports water molecules and is expressed in different cell types throughout the body [1,2,3,4,5,6]. In the central nervous system (CNS), AQP4 is expressed in ependymal cells facing the ventricles, and in the end feet of astrocytes lined along synapses, the perivessel, and subpial brain areas [7,8,9,10]. The distribution of AQP4 in astrocytes in certain parts of the CNS is clearly polarized, where AQP4 is primarily concentrated in the plasma membrane of astrocytic processes in close proximity to blood vessels, the ependymal layer, and pia [8,9]. In distinct osmosensory CNS areas, glial processes show little or even no polarization of the AQP4 distribution [8]; in rat and mouse astrocyte cultures, AQP4 is localized at the plasma membrane and intracellularly [11,12]. Two AQP4 isoforms were described—AQP4a (M1) and AQP4c (M23)—which are alternative transcripts from two different initiating methionine sites [1,13,14,15,16,17]
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