Clearance of activity-evoked K⁺ transients and associated glia cell swelling occur independently of AQP4: A study with an isoform-selective AQP4 inhibitor

The mammalian brain consists of 80% water, which is continuously shifted between different compartments and cellular structures by mechanisms that are, to a large extent, unresolved. Aquaporin 4 (AQP4) is abundantly expressed in glia and ependymal cells of the mammalian brain and has been proposed to act as a gatekeeper for brain water dynamics, predominantly based on studies utilizing AQP4-deficient mice. However, these mice have a range of secondary effects due to the gene deletion. An efficient and selective AQP4 inhibitor has thus been sorely needed to validate the results obtained in the AQP4^-/- mice to quantify the contribution of AQP4 to brain fluid dynamics. In AQP4-expressing Xenopus laevis oocytes monitored by a high-resolution volume recording system, we here demonstrate that the compound TGN-020 is such a selective AQP4 inhibitor. TGN-020 targets the tested species of AQP4 with an IC₅₀ of ~3.5 μM, but displays no inhibitory effect on the other AQPs (AQP1-AQP9). With this tool, we employed rat hippocampal slices and ion-sensitive microelectrodes to determine the role of AQP4 in glia cell swelling following neuronal activity. TGN-020-mediated inhibition of AQP4 did not prevent stimulus-induced extracellular space shrinkage, nor did it slow clearance of the activity-evoked K⁺ transient. These data, obtained with a verified isoform-selective AQP4 inhibitor, indicate that AQP4 is not required for the astrocytic contribution to the K⁺ clearance or the associated extracellular space shrinkage.