Measurement and stoichiometry of bumetanide-sensitive (2Na:1K:3Cl) cotransport in ferret red cells

Abstract

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.