title: Characterization of glial cell K-Cl cotransport.
abstract: BACKGROUND
  The molecular mechanism of K-Cl cotransport (KCC) consists of at least 4 isoforms, KCC 1, 2, 3, and 4 which, in multiple combinations, exist in most cells, including erythrocytes and neuronal cells.
  
  
  METHODS
  We utilized reverse-transcriptase-polymerase chain reaction (RT-PCR) and ion flux studies to characterize KCC activity in an immortalized in vitro cell model for fibrous astrocytes, the rat C6 glioblastoma cell. Isoform-specific sets of oligonucleotide primers were synthesized for NKCC1, KCC1, KCC2, KCC3, KCC4, and also for NKCC1 and actin. K-Cl cotransport activity was determined by measuring either the furosemide-sensitive, or the Cl(-)-dependent bumetanide-insensitive Rb(+) (a K(+) congener) influx in the presence of the Na/K pump inhibitor ouabain. Rb(+) influx was measured at a fixed external Cl concentrations, [Cl(-)](e), as a function of varying external Rb concentrations, [Rb(+)](e), and at a fixed [Rb(+)](e) as a function of varying [Cl(-)](e), and with equimolar Cl replacement by anions of the chaotropic series.
  
  
  RESULTS
  RT-PCR of C6 glioblastoma (C6) cells identified mRNA for three KCC isoforms (1, 3, and 4). NKCC1 mRNA was also detected. The apparent K(m) for KCC-mediated Rb(+) influx was 15 mM [Rb(+)](e), and V(max) 12.5 nmol Rb(+) * mg protein(-1) * minute(-1). The calculated apparent K(m) for external Cl(-) was 13 mM and V(max) 14.4 nmol Rb(+) * mg protein(-1) * minute(-1). The anion selectivity sequence of the furosemide-sensitive Rb(+) influx was Cl(-)>>Br-=NO(3)(-)>I(-)=SCN(-)>>Sfm(-) (sulfamate). Established activators of K-Cl cotransport, hyposmotic shock and N-ethylmaleimide (NEM) pretreatment, stimulated furosemide-sensitive Rb(+) influx. A ñ50% NEM-induced loss of intracellular K(+) was prevented by furosemide.
  
  
  CONCLUSIONS
  We have identified by RT-PCR the presence of three distinct KCC isoforms (1, 3, and 4) in rat C6 glioblastoma cells, and functionally characterized the anion selectivity and kinetics of their collective sodium-independent cation-chloride cotransport activity.
authors.affiliation: Cell Biophysics Group, Wright State University, Dayton, OH 4543, USA.
authors.forename: Kenneth B E
authors.initials: KB
authors.lastname: Gagnon
authors.forename: Norma C
authors.initials: NC
authors.lastname: Adragna
authors.forename: Robert E W
authors.initials: RE
authors.lastname: Fyffe
authors.forename: Peter K
authors.initials: PK
authors.lastname: Lauf
mesh_terms: D000818:Animals; D001483:Base Sequence; D002034:Bumetanide / Q000494:pharmacology; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D005665:Furosemide / Q000494:pharmacology; D007038:Hypotonic Solutions; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012413:Rubidium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters / Q000235:genetics* / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D002034:Bumetanide; D002712:Chlorides; D017931:DNA Primers; D005033:Ethylmaleimide; D005665:Furosemide; D007038:Hypotonic Solutions; D010042:Ouabain; D011188:Potassium; D020033:Protein Isoforms; D012333:RNA, Messenger; D012413:Rubidium; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters; D000096922:K Cl- Cotransporters; C578879:Slc12a2 protein, rat; C492281:Slc12a6 protein, rat; C492283:Slc12a7 protein, rat