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

Jeffery R. Williams, John A Payne

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34 Scopus citations


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 86Rb+ 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-sensitive86Rb+ 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 (pHi) 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 μM) 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 +.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Issue number4 56-4
StatePublished - Oct 2004


  • Ammonium
  • Cesium
  • Cl homeostasis
  • Competitive inhibition

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology


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