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Pathways of K+ movement across the erythrocyte membrane of frog Rana temporaria were studied using 86Rb as a tracer. The K+ influx was significantly blocked by 0.1 mmol.l-1 ouabain (by 30%) and 1 mmol.l-1 furosemide (by 56%) in the red cells incubated in saline at physiological K+ concentration (2.7 mmol.l-1). Ouabain and furosemide had an additive effect on K+ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K+ influx saturated as f(K+)e with apparent Km values for external Ke+ concentration of 0.96 +/- 0.11 and 4.6 +/- 0.5 mmol.l-1 and Vmax of 0.89 +/- 0.04 and 2.8 +/- 0.4 mmol.l cells-1.h-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of Ke+ medium concentration. Total K+ influx was significantly reduced when frog erythrocytes were incubated in NO3- medium. Furosemide did not affect K+ transport in frog red cells in NO3- media. At the same Ke+ concentration the ouabain-furosemide-insensitive K+ influx in Cl- medium was significantly greater than that in NO3- medium. We found no inhibitory effect of 1 mmol.l-1 furosemide on Na+ influx in frog red cells in Cl- medium. K+ loss from the frog erythrocytes in a K(+)-free medium was significantly reduced (mean 58%) after replacement of Cl- with NO3-. Furosemide (0.5 mmol.l-1) did not produce any significant reduction in the K+ loss in both media. The Cl(-)-dependent component of K+ loss from frog red cells was 5.7 +/- 1.2 mmol.l-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

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In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO(3)) has been shown to mediate a large fraction of the total K(+) transport. In the present study, Cl(-)-dependent and Cl(-)-independent K(+) fluxes via frog erythrocyte membranes were investigated as a function of external and internal K(+) ([K(+)](e) and [K(+)](i)) concentration. The dependence of ouabain-resistant Cl(-)-dependent K(+) ((86)Rb) influx on [K(+)](e) over the range 0-20 mm fitted the Michaelis-Menten equation, with an apparent 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(+)](e) above 20 mm in isotonic media significantly reduced the Cl(-)-dependent K(+) influx due to a reciprocal decrease of the external Na(+) ([Na(+)](e)) concentration below 50 mm. Replacing [Na(+)](e) 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(-)-dependent K(+) influx. Moreover, NMDG(+) Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb(+) uptake and K(+) loss from red cells. Cell swelling did not affect the Na(+)-dependent changes in Rb(+) uptake and K(+) loss. In a nominally K(+)(Rb(+))-free medium, net K(+) loss was reduced after lowering [Na(+)](e) below 50 mm. These results indicate that over 50 mm [Na(+)](e) is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K(+), Cl(-)-dependent K(+) loss in K(+)-free media was a linear function of [K(+)](i), 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 frog erythrocytes exhibits a strong asymmetry with respect to transported K(+) ions. The residual, ouabain-resistant K(+) fluxes in NO(3) were only 5-10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K(+) effluxes in isotonic ( approximately 0. 014 hr(-1)) and hypotonic ( approximately 0.022 hr(-1)) media, but cell swelling resulted in a significant increase in the rate constants.

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