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

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 approximately 25% stimulated transport by increasing maximum velocity (Jmax) approximately 1.5-fold and by increasing apparent affinity for external K (Ko) nearly twofold. Dithiothreitol (DTT) was shown to be a partial, reversible inhibitor of K-Cl cotransport. It inhibited in cells of normal volume by reducing Jmax more than twofold; apparent affinity for Ko was increased by DTT, suggesting that DTT stabilizes the transporter-Ko complex. Cell swelling reduced the extent of inhibition by DTT: Jmax 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+, Mgc, stimulated cotransport. Swelling of low-Mg2+ cells stimulated transport further, but only by raising apparent affinity for Ko nearly threefold: Jmax was unaffected. Thus effects of swelling on Jmax and apparent affinity are separable processes. The inhibitory effects of Mgc 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 Ko, low; and Mgc, which affects Jmax, perhaps by holding some transporters in an inactive form. Swelling stimulates transport by relieving both types of inhibition.