Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

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Abstract
86Rb uptake into LLC-PK1 cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na+ and Cl− for activity, and a “leak” component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of86Rb,22Na and36Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na+∶K+∶Cl− cotransport system. The kinetic parameters for Na+ and K+ interaction have been analyzed under initial linear zerotrans conditions. The following values were obtained:K mNa+=0.42±0.05 mmol/liter,V max=303±24 pmol/mg/6 sec;K mK+=11.9±1.0 mmol/liter,V maxK+=307±27 pmol/mg/6 sec. For Cl− interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na+∶1K+∶2Cl− can be calculated. It is concluded that the apical membrane of LLC-PK1 cells contains a Na+∶K+∶2Cl− cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle.

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