Inhibitors of Mammalian Aquaporin Water Channels.

Aquaporins (AQPs) are water channel proteins that are essential to life, being expressed in all kingdoms. In humans, there are 13 AQPs, at least one of which is found in every organ system. The structural biology of the AQP family is well-established and many functions for AQPs have been reported in health and disease. AQP expression is linked to numerous pathologies including tumor metastasis, fluid dysregulation, and traumatic injury. The targeted modulation of AQPs therefore presents an opportunity to develop novel treatments for diverse conditions. Various techniques such as video microscopy, light scattering and fluorescence quenching have been used to test putative AQP inhibitors in both AQP-expressing mammalian cells and heterologous expression systems. The inherent variability within these methods has caused discrepancy and many molecules that are inhibitory in one experimental system (such as tetraethylammonium, acetazolamide, and anti-epileptic drugs) have no activity in others. Some heavy metal ions (that would not be suitable for therapeutic use) and the compound, TGN-020, have been shown to inhibit some AQPs. Clinical trials for neuromyelitis optica treatments using anti-AQP4 IgG are in progress. However, these antibodies have no effect on water transport. More research to standardize high-throughput assays is required to identify AQP modulators for which there is an urgent and unmet clinical need.

Molecular mechanisms governing aquaporin relocalisation.

The aquaporins (AQPs) form a family of integral membrane proteins that facilitate the movement of water across biological membrane by osmosis, as well as facilitating the diffusion of small polar solutes. AQPs have been recognised as drug targets for a variety of disorders associated with disrupted water or solute transport, including brain oedema following stroke or trauma, epilepsy, cancer cell migration and tumour angiogenesis, metabolic disorders, and inflammation. Despite this, drug discovery for AQPs has made little progress due to a lack of reproducible high-throughput assays and difficulties with the druggability of AQP proteins. However, recent studies have suggested that targetting the trafficking of AQP proteins to the plasma membrane is a viable alternative drug target to direct inhibition of the water-conducting pore. Here we review the literature on the trafficking of mammalian AQPs with a view to highlighting potential new drug targets for a variety of conditions associated with disrupted water and solute homeostasis.

Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells.

Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Kitchen and Conner (2015). For the underlying methodology development, please refer to Fenton et al. (2010) and Solenov et al. (2004).