Kinetics of Cl-dependent K influx in human erythrocytes with and without external Na: Effect of NEM

The majority of the ouabain-insensitive K influx in human erythrocytes is dependent on the presence of Cl. Recent studies have shown that a portion of the Cl-dependent K influx persists in the absence of external Na (Na(o)). It has been suggested that this Na(o)-independent component represents (K + Cl) cotransport, whereas the remainder of the Cl-dependent K influx seen on addition of external Na represents (Na + K + 2Cl) cotransport. In the present studies, the kinetics of Cl-dependent K influx were examined in the presence and absence of external Na, by varying external K and external Cl. Our studies suggest that the Na(o)-independent Cl-dependent pathway has a relatively low affinity for external K (K(m) 17-30 mM) in contrast to the high affinity of the Na(o)-augmented component (K(m) 3-4 mM). N-ethylmaleimide (NEM) stimulates the maximal velocity of the Na(o)-independent Cl-dependent K influx achievable without alteration of intracellular solutes but does not alter its K(m) for external K. In contrast, NEM has no stimulatory effect on the Na(o)-agumented component. The Cl dependence of the Na(o)-independent K influx is best described by a relatively flat curve with a mild upward concavity. The kinetic properties of the Na(o)-independent component of Cl-dependent K transport are very similar to those of the putative (K + Cl) cotransport pathway seen in low-K sheep erythrocytes. When the Cl-dependent K influx is measured in high-Na media, the meausred K(m) for K(o) is a consequence of the individual K(m) values and the relative magnitudes of the low-affinity Na(o)-independent component and the relatively high-affinity Na(o)-augmented component.