Kinetics of Cl-dependent K fluxes in hyposmotically swollen low K sheep erythrocytes

A detailed kinetic study of K:Cl cotransport in hyposmotically swollen low K sheep red blood cells was carried out to characterize the nature of the outwardly poised carrier. The kinetic parameters were determined from the rate of K efflux and influx under zero-K-trans conditions in red cells with cellular K altered by the nystatin method and with different extracellular K or Rb concentrations. Although apparent affinities for efflux and influx were quite similar, the maximal velocity for K efflux was approximately two times greater than for influx. Furthermore, at thermodynamic equilibrium (i.e., when the ion product of K and Cl within the cell was equal to that outside) a temperature-dependent net K efflux was observed, approaching zero only when the external product reached approximately two times the internal product. The binding order of the ions to the transporter was asymmetric, being ordered outside (Cl binding first, followed by K) and random inside. K efflux but not influx was trans-inhibited by KCl. Trans inhibition of K efflux was used to verify the order of binding outside: trans inhibition by external Cl occurred in the absence of external K, but not vice versa. Thus K:Cl cotransport is kinetically asymmetric in hyposmotically swollen low K sheep red cells.