Functional characterization of the neuronal-specific K-Cl cotransporter: Implications for [K⁺](o) regulation

The neuronal K-Cl cotransporter isoform (KCC2) was functionally expressed in human embryonic kidney (HEK-293) cell lines. Two stably transfected HEK-293 cell lines were prepared: one expressing an epitope- tagged KCC2 (KCC2-22T) and another expressing the unaltered KCC2 (KCC2-9). The KCC2-22T cells produced a glycoprotein of ~150 kDa that was absent from HEK-293 control cells. The ⁸⁶Rb influx in both cell lines was significantly greater than untransfected control HEK-293 cells. The KCC2-9 cells displayed a constitutively active ⁸⁶Rb influx that could be increased further by i mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant (K(i)) ~25 μM] and bumetanide (K(i) ~55 μM) inhibited the NEM- stimulated ⁸⁶Rb influx in the KCC2-9 cells. This diuretic-sensitive ⁸⁶Rb influx in the KCC2-9 cells, operationally defined as KCC2 mediated, required external Cl^- but not external Na⁺ and exhibited a high apparent affinity for external Rb⁺(K⁺) [Michaelis constant (K(m)) = 5.2 ± 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl^- (K(m) >50 mM). On the basis of thermodynamic considerations as well as the unique kinetic properties of the KCC2 isoform, it is hypothesized that KCC2 may serve a dual function in neurons: 1) the maintenance of low intracellular Cl^- concentration so as to allow Cl^- influx via ligand-gated Cl^- channels and 2) the buffering of external K⁺ concentration ([K⁺](o) in the brain.