title: Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation.
abstract: 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 approximately 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 1 mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant (Ki) approximately 25 microM] and bumetanide (Ki approximately 55 microM) 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 (Km) = 5.2 +/- 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl- (Km > 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.
authors.affiliation: Department of Human Physiology, University of California, School of Medicine, Davis 95616, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Payne
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001921:Brain / Q000378:metabolism*; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D005033:Ethylmaleimide / Q000494:pharmacology; D005665:Furosemide / Q000494:pharmacology; D006706:Homeostasis; D006801:Humans; D007038:Hypotonic Solutions; D007668:Kidney; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012413:Rubidium / Q000493:pharmacokinetics; D012414:Rubidium Radioisotopes; D027981:Symporters; D013816:Thermodynamics; D014162:Transfection; D000096922:K Cl- Cotransporters
chemical_list: D002034:Bumetanide; D002352:Carrier Proteins; D005033:Ethylmaleimide; D005665:Furosemide; D007038:Hypotonic Solutions; D011188:Potassium; D011994:Recombinant Proteins; D012413:Rubidium; D012414:Rubidium Radioisotopes; D027981:Symporters; D000096922:K Cl- Cotransporters