Keeping cells in shape

Cell size is determined by the balance of positively and negatively charged ions and other solutes in the fluids inside and outside cells.  The physical process of osmosis dictates that if the solute concentrations inside and outside the cell are different, water will tend to move across the cell membrane from the side with low solute concentration (hypotonic) to the side with high solute concentration (hypertonic).  Without any additional controls, cells would be vulnerable to changes in solute concentration, dramatically shrinking after a salt load or swelling after a water load.

To avoid such dangerous abrupt shifts in volume, cells need to rapidly adjust their internal solute concentration to compensate for external changes.  They accomplish this principally by altering the entry/exit balance of chloride ions. Chloride exits the cell via potassium-chloride cotransporters (KCC)and enters the cell via the sodium-potassium-chloride cotransporter (NKCC1).  The regulation of these two complementary cotransporters is coordinated.  Phosphorylation, addition of phosphate groups to the cotransporters, activates NKCC1 while inhibiting KCC, whereas dephosphorylation, removal of phosphate groups, has the opposite effect.

Until now, specific sites of phosphorylation in KCC had not been identified.  Fondation Leducq-supported investigators Drs. Jesse Rinehart and Richard Lifton, of the Transatlantic Network on Hypertension, along with colleagues from Yale University and the University of Cincinnati College of Medicine, applied innovative techniques to discover the KCC regulatory sites.  These findings, which may have implications in areas as diverse as sickle cell anemia and neurologic disorders, were published in the August 7, 2009 issue of Cell.

The investigators generated recombinant human KCC proteins, which they digested into smaller peptide fragments.  They then used a special matrix to selectively isolate only the phosphorylated peptide fragments.  They obtained 5 fragments, for which they determined the amino acid sequence by mass spectrometry techniques.  The next step was to determine whether any of these 5 sites underwent changes in phosphorylation in response to different ionic conditions.  Using a special isotope labeling technique, the investigators were able to monitor the status of phosphorylation at these sites in cells exposed to either normal or hypotonic conditions.  They found that hypotonic conditions reproducibly caused dephosphorylation of KCC at 2 of the sites, thereby leading to activation of the cotransporter.

The investigators next hypothesized that keeping these 2 sites dephosphorylated would result in continuous activation of the cotransporter.  They generated mutations in KCC that made phosphorylation impossible at the 2 sites.  Mutation of each site individually caused KCC to be more active, even in isotonic conditions when normal KCC has little activity.  When both sites were mutated, the effect was synergistic, resulting in activity levels 25 times greater than normal.  Cells bearing the 2 mutations shrank even in isotonic conditions, and began to die after 16 hours.

The potential broad implications of this discovery was underscored by the finding that the amino acid sequences at the 2 sites were identical in 11 animal species that were examined, including mouse and fish.

KCC is the major transport mechanism in human red blood cells.  KCC is normally inactive in isotonic conditions, and the investigators confirmed that the two sites are quickly dephosphorylated upon exposure to hypotonic conditions.  In sickle cell anemia, KCC is overactive, leading to dehydration and sickling of the red blood cells. Regulation of chloride ions is also a key component of the response of nerve cells to GABA, a chemical signal that governs alertness that has been implicated in anxiety and other disorders.  KCC activity in the brain also appears to change with age, having little activity in the neonates and high activity in adults.  Knowledge of the 2 KCC regulatory sites thus could provide new strategies for studying and treating sickle cell anemia and neurologic disorders.

Dr. Lifton is an investigator of the Howard Hughes Medical Institute.  This work was also supported by the National Heart, Lung, and Blood Institute; the National Institute on Drug Abuse; the National Center for Research Resources; and the Cincinnati Comprehensive Sickle Cell Center.

Click on the title to access the article in Cell:  Sites of regulated phosphorylation that control K-Cl cotransporter activity.