NKCC1 cotransporters: keeping an ‘ion’ them

The sodium–potassium–chloride cotransporter NKCC1 has a track record of bringing the ions it is named after into the cell. But recently, scientists discovered that the cotransporter brings a substantial amount of water into the cell as well.

In a recent issue of The Journal of Physiology, Hamann et al. (2010) analysed cell volumes of human pigmented epithelial cells from the ciliary body of the eye, which contain NKCC1 at high levels, which pumps sodium, postassium and chloride into the cell. This epithelium produces the aqueous humour that is responsible for the maintenance of proper intra-ocular pressure and is the only nourishment of the cornea and lens. The aqueous humour is constantly replenished, and its composition requires chloride and water to be transported from the blood to the anterior chamber of the eye (Layne et al. 2001).

Steffen Hamann and colleagues at the University of Copenhagen, the Instituto de Psiquiatría in Mexico City, and Wright State University in Dayton, Ohio wondered why it seems that cell volumes in pigmented epithelial cells depend on the activity of NKCC1. They found that it is because the cotransporter actively brings water into the cell (Hamann et al. 2010).

The investigators had previously found that in addition to its known transport of sodium, potassium, and chloride ions into the cell, NKCC1 seemed to be permeable to water (Hamann et al. 2005). However, it was not known whether this was active or passive transport, and whether its transport was stoichiometrically linked to the cotransporter.

By culturing these pigmented epithelial cells in a monolayer and imaging intracellular calcein fluorescence, the group could determine cell volume at any point as the solutions bathing the cells were rapidly changed. The rate of cell volume changes during a solution change determined the flux of water across the baso-lateral membranes of these cells.

In vivo, there is an osmotic gradient for chloride and water to pass from blood to the anterior chamber through the pigmented epithelium. This study therefore abrogated the gradient by culturing the cells and keeping the solutions isosmotic, while changing the concentration of the ions that NKCC1 transports. In this way, the changes in water flux would directly reflect the role of NKCC1 when changing the bath solutions. Water flux into the cell was stopped when sodium or chloride was removed abruptly from the solution and replaced with electrically equivalent non-permeant molecules to preserve osmolarity. To further show that this flux was due to NKCC1, the investigators added bumetanide or calyculin-A during these changes (an inhibitor and an activator of the cotransporter) and the flux of water was respectively inhibited or stimulated.

But the most interesting finding was that NKCC1 transports water uphill, against an osmotic gradient. The team had observed that adding mannitol to increase the osmolarity of the solution caused a net flux of water out of the cell. Since chloride is required for NKCC1 activity, they then measured the flux of water as they added chloride to a previously chloride-free solution (which should activate NKCC1), while simultaneously adding mannitol (which should cause water to flow out of the cell). The team observed that water in fact flowed into the cell, against an osmotic gradient. In this way, they were able to show that water is actively transported by NKCC1 into the cell.

In fact, from calculations of water volume changes and ion concentrations during the experiments, it is estimated that for every NKCC1 transport cycle, 570 water molecules are cotransported with 1 sodium ion, 1 potassium ion, and 2 chloride ions! The ability for the pigmented epithelium to cotransport water away from the blood and toward the anterior chamber of the eye could be important in ensuring that aqueous humour is consistently made, regardless of the osmolarity of the blood.

Of course, knowing that sodium, potassium, chloride and water are transported into the pigmented epithelium, but that there is less sodium and potassium in the aqueous humour, points to a discrepancy in NKCC1 as being the only channel or transporter involved in the production of the fluid. The authors suggest that the large amount of sodium–potassium ATPases on the baso-lateral side of the pigmented epithelium (the same side as NKCC1) allows for sodium to be transported out of the cell. Though the intracellular concentrations of potassium in cells is usually high, for there to be a constant influx of potassium by NKCC1 and Na⁺,K⁺-ATPases without a way for potassium to flow out of the cell and into the blood means that this explanation is not sufficient, or that there needs to be a better investigation of potassium channels at the baso-lateral surface of these cells, if that is truly the mechanism for ion transport in this epithelium.

In addition, the proposal of water cotransport by NKCC1 as a primary means for producing aqueous humour produces new questions that are rather far from the scope of the study, but nonetheless merit some discussion. The foremost of these comes from a simple calculation. If we were to consider each cycle of transport to be an influx of a ‘solution’ of ions, their concentrations would be on the order of 0.1 to 0.2 m. These are concentrations that range from two to three orders of magnitude above the physiological concentrations of ions inside the cell, in the blood, and in the aqueous humour. In particular, if NKCC1 is the primary means for chloride and water transport into the aqueous humour, then the cell is either pumping too much chloride across the epithelium, or too little water. Given that ion transport can be energetically costly, that there are other means for passive water transport, and that there is an osmotic gradient for water to pass from blood to the anterior chamber indicates that NKCC1 is probably not the primary means for water transport at this epithelium. It is far more likely that passive water transport moves most water into the anterior chamber. However, the existence of a cotransporter that will bring water into the cell regardless of the osmolarity of the extracellular solution could be a ‘safety net’ in case blood becomes too hypertonic for passive water transport to occur efficiently.

Thanks to this study, the discovery of stoichiometric cotransport of water by NKCC1 is clear, and it is in line with reports of water cotransport by other molecules, such as MCT1 (a proton–lactate cotransporter). However, osmosis can also take place in other water cotransporters, such as SGLT1 (a sodium–glucose cotransporter) and EAAT1 (a sodium–proton–potassium–glutamate cotransporter) (Zeuthen, 2010). This difference in mechanism further motivates the question of physiological significance: precisely what function do these cotransporters have, not only in the pigmented epithelium of the ciliary body of the eye, but also in the other epithelia arranged throughout the body?