Measurement and stoichiometry of bumetanide-sensitive (2Na:1K:3Cl) cotransport in ferret red cells.
The bumetanide-sensitive uptake of Na+, K+(Rb+) and Cl- has been measured at 21 degrees C in ferret red cells treated with (SITS + DIDS) to minimize anion flux via capnophorin (Band 3). During the time course of the influx experiments tracer uptake was a first-order rate process. At normal levels of external Na+ (150 mM) the bumetanide-sensitive uptake of K+ was dependent on Cl- and represented almost all of the K+ uptake, the residual flux demonstrating linear concentration dependence. The uptake of Na+ and Cl- was only partially inhibited by bumetanide indicating that pathways other than (Na + K + Cl) cotransport participate in these fluxes. The diuretic-sensitive uptake of Na+ or Cl- was, however, abolished by the removal of K+ or the complementary ion indicating that bumetanide-sensitive fluxes of Na+, K+ and Cl- are closely coupled. At very low levels of [Na]o (less than 5 mM) K+ influx demonstrated complex kinetics, and there was evidence of the unmasking of a bumetanide-sensitive Na+-independent K+ transport pathway. The stoichiometry of bumetanide-sensitive tracer uptake was 2Na:1K:3Cl both in cells suspended in a low and a high K+-containing medium. The bumetanide-sensitive flux was markedly reduced by ATP depletion. We conclude that a bumetanide-sensitive cotransport of (2Na:1K:3Cl) occurs as an electroneutral complex across the ferret red cell membrane.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology*; D002712:Chlorides / Q000097:blood*; D004232:Diuretics / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005289:Ferrets; D007700:Kinetics; D011188:Potassium / Q000097:blood*; D012964:Sodium / Q000097:blood*
D002712:Chlorides; D004232:Diuretics; D002034:Bumetanide; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium