Characterization of a Na : K : 2C1 cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

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 of 86Rb, 22Na and 36Cl 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 zero trans conditions. The following values were obtained KmNa+ = 0.42 +/- 0.05 mmol/liter, Vmax = 303 +/- 24 pmol/mg/6 sec; KmK+ = 11.9 +/- 1.0 mmol/liter, VmaxK+ = 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- co-transport system with properties similar to those described for the thick ascending limb of the loop of Henle.