Kinetics of K-Cl cotransport in frog erythrocyte membrane: Effect of external sodium

In frog red blood cells, K-Cl cotransport (i.e., the difference between oua bain-resistant K fluxes in Cl and NO3) has been shown to mediate a large fr action of the total K+ transport. In the present study, Cl--dependent and C l--independent K+ fluxes via frog erythrocyte membranes were investigated a s a function of external and internal K+ ([K+](e) and [K+](i)) concentratio n. The dependence of ouabain-resistant Cl--dependent K+ (Rb-86) influx on [ K+], over the range 0-20 mM fitted the Michaelis-Menten equation, with an a pparent affinity (K-m) of 8.2 +/- 1.3 mM and maximal velocity (V-max) of 10 .4 +/- 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl--dependent K+ influx increased both K-m (12.8 +/- 1.7 mM, P < 0. 05) and V-max (20.2 +/- 2.9 mmol/l/hr, P < 0.001). Raising [K+], above 20 m M in isotonic media significantly reduced the Cl--dependent K+ influx due t o a reciprocal decrease of the external Na+ ([Na+],) concentration below 50 mM. Replacing [Na+], by NMDG(+) markedly decreased V-max (3.2 +/- 0.7 mmol /l/hr, P < 0.001) and increased K-m (15.7 +/- 2.1 mM, P < 0.03) of Cl--depe ndent K+ influx. Moreover, NMDG(+) Cl substitution for NaCl in isotonic and hypotonic media containing 10 mM RbCl significantly reduced both Rb+ uptak e and K+ loss from red cells. Cell swelling did not affect the Na+-dependen t changes in Rb+ uptake and K+ loss. In a nominally K+(Rb+)-free medium, ne t K+ loss was reduced after lowering [Na+](e) below 50 mM. These results in dicate that over 50 mM [Na+](e) is required for complete activation of the K-CI cotransporter. In nystatin-pretreated cells with various intracellular K+, Cl--dependent K+ loss in K+-free media was a linear function of [K+], with a rate constant of 0.11 +/- 0.01 and 0.18 +/- 0.008 hr(-1) (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in fr og erythrocytes exhibits a strong asymmetry with respect to transported Kions. The residual, ouabain-resistant K+ fluxes in NO3 were only 5-10% of t he total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K+ effluxes in iso tonic (similar to 0.014 hr(-1)) and hypotonic (similar to 0.022 hr(-1)) med ia, but cell swelling resulted in a significant increase in the rate consta nts.