Effect of 1-chloro-2,4-dinitrobenzene on K⁺ transport in normal and sickle human red blood cells

1-Chloro-2,4-dinitrobenzene (CDNB), which causes oxidative stress through depletion of reduced glutathione (GSH), increases the passive K⁺ permeability of red cells. In this paper, we investigated the effects of CDNB (1 mM) on the activities of the K⁺-Cl^- cotransporter (KCC; measured as Cl^--dependent K⁺ influx) and the Gardos channel (taken as clotrimazole-sensitive K⁺ influx, 5 μM) in human red cells, using ⁸⁶Rb⁺ as a K⁺ congener. ⁴⁵Ca²⁺ was used to study passive Ca²⁺ entry and active Ca²⁺ efflux via the plasma membrane Ca²⁺ pump. Both the Gardos channel and KCC were stimulated in both normal and sickle red cells. In sickle cells, stimulation of KCC was similar in oxygenated and deoxygenated cells; that of the Gardos channel was greater in deoxygenated cells. In normal red cells, stimulation of both pathways was greater in oxygenated cells (by 4 ± 1-fold; all means ± S.E.M., n = 3). The effects on the Gardos channel were dependent on extracellular Ca²⁺ and were associated with inhibition of the plasma membrane Ca²⁺ pump (by 29 ± 3 %, P < 0.01) and increased Ca²⁺ sensitivity of the channel (EC₅₀ for [Ca²⁺]_i reduced from 260 ± 26 to 175 ± 15 nM; P < 0.05). Cell volume, pH_i, ATP levels and passive Ca²⁺ entry were not affected by CDNB. The effects on KCC were inhibited (93 ± 6 %) by prior treatment with the protein phosphatase inhibitor calyculin A (100 nM) and were not additive with stimulation by N-ethylmaleimide (1 mM), regardless of the order of addition. These findings are therefore consistent with inhibition of a regulatory protein kinase, although stimulation of the conjugate protein phosphatase(s) may also occur. KCC stimulation was also Ca²⁺ dependent. These findings are important for understanding how GSH depletion alters membrane permeability and how to protect against red cell dehydration.