Abstract
Red blood cells of several species are known to exhibit a ouabain-insensitive, anion-dependent K+ (Rb+) flux that is stimulated by cell swelling. We have used rabbit red cells to study the kinetics of activation and inactivation of the flux upon step changes in tonicity. Sudden hypotonic swelling (210 mosmol) activates the flux after a lag period of 10 min at 37 degrees C and 30-50 min at 25 degrees C. In cells that were preswollen to activate the transporter, sudden shrinkage (by addition of hypertonic NaCl) causes a rapid inactivation of the flux; the time lag for inactivation is less than 2 min at 37 degrees C. A minimal model of the volume-sensitive KCl transport system requires two states of the transporter. The activated (A) state catalyzes transport at some finite rate (turnover number unknown because the number of transporters is unknown). The resting (R) state has a much lower or possibly zero transport rate. The interconversion between the states is characterized by unimolecular rate constants R k12 in equilibrium with k21 A. The rate of relaxation to any new steady state is equal to the sum of the rate constants k12 + k21. Because the rate of transport activation in a hypotonic medium is lower than the rate of inactivation in an isotonic medium, we conclude that the volume-sensitive rate process is inactivation (the A to R transition); that is, cell swelling activates transport by lowering k21. Three phosphatase inhibitors (fluoride, orthovanadate, and inorganic phosphate) all inhibit the swelling-activated flux and also slow down the rate of approach to the swollen steady state. This finding suggests that a net dephosphorylation is necessary for activation of the flux and that the net dephosphorylation takes place as a result of swelling-induced inhibition of a kinase rather than stimulation of a phosphatase.