A special role of Na,K-ATPaseand its molecular partners for astrocyte function

Sammanfattning: Astrocytes are glial cells that express several specific transporters and channels with specialized functions to maintain water, ion and neurotransmitter concentrations in order to preserve normal neuronal function. Astrocytes express two catalytic isoforms of plasma membrane enzyme Sodium Potassium-ATPase (Na,K-ATPase), that are essential for several of astrocyte’s functions. In each cycle Na,K-ATPase actively transports 3 Na+ ions out of the cell and 2 K+ ions into the cell, using the energy of one ATP molecule. Na,K-ATPase establishes transmembrane Na+ gradient that allows for efficient Na+-coupled transport, including glutamate uptake. Na,K-ATPase is also responsible for active K+ uptake from extracellular space and therefore maintenance of extracellular K+ homeostasis. The overall goal of this thesis was to study the molecular and functional interactions between the Na,K-ATPase and other proteins responsible for astrocyte function. The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. Emerging evidence suggests that AQP4 facilitates extracellular K+ clearance by astrocytes and contributes to recovery of neuronal excitability. We found that AQP4 can assemble with its regulator metabotropic glutamate receptor 5 (mGluR5) and with Na,K-ATPase. The AQP4 NH2-terminus was shown to interact with Na,K-ATPase catalytic ? subunit and with mGluR5. Föster resonance energy transfer (FRET) studies in primary astrocytes derived from rat striatum showed that interaction occurs in intact cells. Thus AQP4/Na,K- ATPase/mGluR5 can form a macromolecular complex in astrocytes, that may be of functional importance for the regulation of water and K+ homeostasis in the brain. Astrocytes express two isoforms of the Na,K-ATPase catalytic ? subunit: the ubiquitous ?1 and the ?2, which in the brain is predominantly expressed in astrocytes. The ?2 isoform has lower Na+ affinity than ?1. We explored the relative roles of the ?1 and ?2 isoforms for the support of Na+-coupled glutamate uptake in primary astrocytes. We found that the ?2 isoform contributes to a more efficient restoration of increases in intracellular sodium concentration ([Na+]i) evoked by the Na+-coupled glutamate uptake. Both ?1 and ?2 interacted molecularly with glutamate transporters via the 1st intracellular loop, but the interaction with ?2 appeared stronger. The study points to a specific role for ?2 in the handling of [Na+]i transients in astrocytes and suggests that ?1 and ?2 may differ with regard to capacity to interact with the glutamate transporters. Mutations in the Na,K-ATPase ?2 subunit are associated with the neurological disease familial hemiplegic migraine type 2 (FHM2). In this study we ii determined ?1 and ?2 abundance and glutamate uptake in primary cultures from heterozygous and homozygous ?2 mutant mice carrying the human knock-in FHM2-mutation G301R. Both ?2 abundance and glutamate uptake were significantly reduced in astrocytes expressing the mutant ?2. The plasma membrane expression of mutant Venus-tagged ?2 was also reduced in comparison to wild type ?2. The results suggest that reduced capacity of astrocytes expressing ?2 G301R mutant to take up glutamate, may lead to temporary increases in ambient glutamate concentration that, at least to some extent, may contribute to the symptoms in FHM2.