Cation transport by the neuronal K+-Cl- cotransporter KCC2:: thermodynamics and kinetics of alternate transport modes

Both Cs+ and NH4+ alter neuronal Cl- homeostasis, yet the mechanisms have not been clearly elucidated. We hypothesized that these two cations altered the operation of the neuronal K+-Cl- cotransporter (KCC2). Using exogenously expressed KCC2 protein, we first examined the interaction of cations at the transport site of KCC2 by monitoring furosemide-sensitive Rb-86(+) influx as a function of external Rb+ concentration at different fixed external cation concentrations (Na+, Li+, K+, Cs+, and NH4+). Neither Na+ nor Li+ affected furosemide-sensitive Rb-86(+) influx, indicating their inability to interact at the cation translocation site of KCC2. As expected for an enzyme that accepts Rb+ and K+ as alternate substrates, K+ was a competitive inhibitor of Rb+ transport by KCC2. Like K+, both Cs+ and NH4+ behaved as competitive inhibitors of Rb+ transport by KCC2, indicating their potential as transport substrates. Using ion chromatography to measure unidirectional Rb+ and Cs+ influxes, we determined that although KCC2 was capable of transporting Cs+, it did so with a lower apparent affinity and maximal velocity compared with Rb+. To assess NH4+ transport by KCC2, we monitored intracellular pH (pH(i)) with a pH-sensitive fluorescent dye after an NH4+-induced alkaline load. Cells expressing KCC2 protein recovered pHi much more rapidly than untransfected cells, indicating that KCC2 can mediate net NH4+ uptake. Consistent with KCC2-mediated NH4+ transport, pHi recovery in KCC2-expressing cells could be inhibited by furosemide (200 muM) or removal of external [Cl-]. Thermodynamic and kinetic considerations of KCC2 operating in alternate transport modes can explain altered neuronal Cl- homeostasis in the presence of Cs+ and NH4+.