Cation transport by the neuronal K(+)-Cl(-) cotransporter KCC2: thermodynamics and kinetics of alternate transport modes.
Both Cs(+) and NH(4)(+) 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 (86)Rb(+) influx as a function of external Rb(+) concentration at different fixed external cation concentrations (Na(+), Li(+), K(+), Cs(+), and NH(4)(+)). Neither Na(+) nor Li(+) affected furosemide-sensitive (86)Rb(+) 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 NH(4)(+) 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 NH(4)(+) transport by KCC2, we monitored intracellular pH (pH(i)) with a pH-sensitive fluorescent dye after an NH(4)(+)-induced alkaline load. Cells expressing KCC2 protein recovered pH(i) much more rapidly than untransfected cells, indicating that KCC2 can mediate net NH(4)(+) uptake. Consistent with KCC2-mediated NH(4)(+) transport, pH(i) recovery in KCC2-expressing cells could be inhibited by furosemide (200 microM) 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 NH(4)(+).
Williams
Jeffery R
JR
Department of Physiology and Membrane Biology, One Shields Ave., University of California, Davis, CA 95616-8644, USA.
Payne
John A
JA
D000818:Animals; D002412:Cations / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002586:Cesium / Q000737:chemistry / Q000378:metabolism; D002713:Chlorine / Q000737:chemistry / Q000378:metabolism; D004285:Dogs; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry; D017136:Ion Transport / Q000502:physiology*; D007700:Kinetics; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D012414:Rubidium Radioisotopes / Q000737:chemistry / Q000378:metabolism; D027981:Symporters / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D000096922:K Cl- Cotransporters
D002412:Cations; D002586:Cesium; D002713:Chlorine; D000644:Quaternary Ammonium Compounds; D012414:Rubidium Radioisotopes; D027981:Symporters; D000096922:K Cl- Cotransporters