K-Cl cotransport in LK sheep erythrocytes: Kinetics of stimulation by cell swelling

Summary The effects of osmotic cell swelling were studied on the kinetics of Cl-dependent K⁺ influx, K–Cl cotransport, in erythrocytes from sheep of the low K⁺ (LK) phenotype. Swelling 25% stimulated transport by increasing maximum velocity (J_max) 1.5-fold and by increasing apparent affinity for external K (K_o) nearly twofold. Dithiothreitol (DTT) was shown to be a partial, reversible inhibitor of K–Cl cotransport. It inhibited in cells of normal volume by reducingJ_max more than twofold: apparent affinity for K_o was increased by DTT, suggesting that DTT stabilizes the transporter-K_o complex. Cell swelling reduced the extent of inhibition by DTT:J_max was inhibited by only about one-third in swollen cells, and apparent affinity was only slightly affected. This result suggested that DTT does not act directly on the transporter, but on a hypothetical regulator, an endogenous inhibitor. Swelling relieves inhibition by the regulator, and reduces the effect of DTT. Reducing intracellular Mg²⁺, Mg_o, stimulated cotransport. Swelling of low-Mg²⁺ cells stimulated transport further, but only by raising apparent affinity for K_o nearly threefold:J_max was unaffected. Thus effects of swelling onJ_max and apparent affinity are separable processes. The inhibitory effects of Mg_o and DTT were shown to be additive, indicating separate modes of action. There appear to be two endogenous inhibitors: the hypothetical regulator, which holds affinity for K_o, low; and Mg_o, which affectsJ_max perhaps by holding some transporters in an inactive form. Swelling stimulates transport by relieving both types of inhibition.