Cotransport of water by the Na+–K+–2Cl− cotransporter NKCC1 in mammalian epithelial cells

Water transport by the Na+–K+–2Cl− cotransporter (NKCC1) was studied in confluent cultures of pigmented epithelial (PE) cells from the ciliary body of the fetal human eye. Interdependence among water, Na+ and Cl− fluxes mediated by NKCC1 was inferred from changes in cell water volume, monitored by intracellular self‐quenching of the fluorescent dye calcein. Isosmotic removal of external Cl− or Na+ caused a rapid efflux of water from the cells, which was inhibited by bumetanide (10 μm). When returned to the control solution there was a rapid water influx that required the simultaneous presence of external Na+ and Cl−. The water influx could proceed uphill, against a transmembrane osmotic gradient, suggesting that energy contained in the ion fluxes can be transferred to the water flux. The influx of water induced by changes in external [Cl−] saturated in a sigmoidal fashion with a Km of 60 mm, while that induced by changes in external [Na+] followed first order kinetics with a Km of about 40 mm. These parameters are consistent with ion transport mediated by NKCC1. Our findings support a previous investigation, in which we showed water transport by NKCC1 to be a result of a balance between ionic and osmotic gradients. The coupling between salt and water transport in NKCC1 represents a novel aspect of cellular water homeostasis where cells can change their volume independently of the direction of an osmotic gradient across the membrane. This has relevance for both epithelial and symmetrical cells. Cell volume control is fundamental for cell survival. Cells have evolved mechanisms for maintaining their volume constant. These mechanisms involve the movement of solutes and water across the plasma membrane through specialized proteins. The water within a cell ultimately determines its volume and has been assumed to cross the cell membrane exclusively through channels called aquaporins. We show that water also crosses the membrane carried by NKCC1, a membrane protein belonging to the Na+–K+–Cl− cotransporter (NKCC) family. This membrane protein transports 1 sodium, 1 potassium and 2 chloride ions together with a large number of water molecules per cycle. A key finding is that NKCC1 transports water uphill, against an osmotic gradient. These observations increase our knowledge of how cells and tissues handle water, and are important for understanding medical conditions like brain oedema, intracranial hypertension, glaucoma and airway hydration disorders.