Functional and molecular interactions between aquaporins and Na,K-ATPase

Abstract The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes and provides a mechanism by which water permeability of the plasma membrane can be regulated. Astrocytes play a key role in the clearance of both potassium (K+ ) and glutamate released during neuronal activity. Emerging evidence suggests that AQP4 facilitates K+ clearance by astrocytes and contributes to recovery of neuronal excitability. Here we report that AQP4 can assemble with its regulator metabotropic glutamate receptor 5 (mGluR5) and with Na,K-ATPase; the enzyme responsible for active K+ transport and for establishing the electrochemical gradient across the cell plasma membrane. We have, by use of pull down assays in rat brain tissue, identified the segment in the AQP4 NH2 -terminus containing the amino acid residues 23–32 as the site for interaction with Na,K-ATPase catalytic subunit and with mGluR5. Mutagenesis studies revealed that the AQP4 amino acids K27 and W30 are of key importance for interaction with both Na,K-ATPase and mGluR5. To confirm that interaction also occurs within intact cells, we have performed fluorescence resonance energy transfer (FRET) studies in primary astrocytes derived from rat striatum. The results indicate close proximity of wild type AQP4 and Na,K-ATPase in the plasma membrane of rat astrocytes. FRET efficiencies observed with the mutants AQP4 K27A and AQP4 W30A were significantly lower, highlighting the importance of these residues for the interaction between AQP4 and Na,K-ATPase. We conclude that AQP4/Na,K-ATPase/mGluR5 can form a macromolecular complex/transporting microdomain in astrocytes. This complex may be of functional importance for the regulation of water and K+ homeostasis in the brain, as well as for neuron-astrocyte metabolic crosstalk.