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

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Abstract
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.

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