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

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Abstract
The bumetanide-sensitive uptake of Na+, K−(Rb−) and Cl− has been measured at 21°C in ferrent 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+ (150mm) 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 (+ 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.