Temperature-induced functional deocclusion of thiols inhibitory for sheep erythrocyte K-Cl cotransport

In low-K sheep erythrocytes, K-Cl cotransport is activated by treatment with low concentrations of thiol reagents and by other interventions such as lowering of cellular free cytosolic Mg, hyposmotic cell swelling, the kinase inhibitor staurosporine, and hydroxylamine. High concentrations of N-ethylmaleimide or methylmethane thiolsulfonate reverse the activation through thiol groups and, as shown here, also the stimulation by non-thiol manipulations. The overriding inhibitory sites functionally associated with and different from those of the activating thiols (SHa) were further distinguished by temperature. Treatment with N-ethylmaleimide and its subsequent removal by dithiothreitol, both at 0 degrees C, prevented the inhibitory effect at 37 degrees C and thus the chemical modification of inhibitory thiols (SHi). Whereas stimulation through SHa closely followed the loss of glutathione, inhibition through SHi occurred only in glutathione-depleted cells. The reversal of K-Cl cotransport stimulation by all hitherto known interventions, which is strongest in metabolically depleted cells, suggests that the low temperature-protected SHi constitute crucial sites that, close to the transporter itself or at the cytoskeletal level, become functionally deoccluded upon temperature elevation.