The neuronal-specific K-C1 cotransporter (KCC2) exhibits high apparent affinity for external K⁺: Implications for external K⁺ buffering in the brain

A unique isoform of the K-C1 cotransporter (KCC2) has been identified in our lab that is expressed specifically in neurons throughout the brain. We have functionally expressed KCC2 in a stable HEK cell line (KCC2-9) and characterized the ouabahvinsensitive ⁸⁶Rb influx in these cells. The KCC2-9 cells exhibited a significant constitutively active 86Rb influx that could be increased further by ImM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide (K_i≈25μM) and bumetanide (K_i=55μM) dramatically inhibited the NEM-stimulated ⁸⁶Rb influx in the KCC2-9 cells. This diuretic-sensitive ⁸⁶Rb influx, which we operationally defined as KCC2-mediated, exhibited a low apparent affinity for external Cl^- (gluconate replacement; K_m>50mM); however, it displayed a remarkably high apparent affinity for external K⁺ (K_m=5.2±0.9mM; mean±SEM, n=5). This external K⁺ affinity is at least 5 times greater than that previously reported for the ubiquitously expressed KCC1 isoform. We have previously proposed that KCC2 is involved in postsynaptic inhibition by maintaining low [Cl^-], (i.e. E_CI<E_m) for the proper function of ligand-gated Cl^- channels. As such, most neurons have low [Cl^-]_i (<10mM), and therefore, K-Cl cotransport is very near thermodynamic equilibrium. Consequently, modest activity-dependent increases in [K⁺]_o, which are common in the brain, will reverse the force driving net K-Cl cotransport from outward to inward. Based on thermodynamic considerations as well as the unique kinetic properties of the KCC2 isoform, we hypothesize that KCC2 can buffer increases in [K⁺]_o, and this function can significantly modulate neuronal activity as KCC2-dependent K⁺ buffering will shift E_Ci to more positive values.