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 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.
Gusev
G P
GP
Sechenov Institute of Evolutionary Physiology and Biochemistry, Thorez pr.44, 194223, St. Petersburg, Russia.
Agalakova
N I
NI
Lapin
A V
AV
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009761:Nystatin / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D011896:Rana temporaria; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D027981:Symporters; D000096922:K Cl- Cotransporters
D002352:Carrier Proteins; D002712:Chlorides; D009761:Nystatin; D010042:Ouabain; D011188:Potassium; D012413:Rubidium; D012964:Sodium; D027981:Symporters; D000096922:K Cl- Cotransporters