1-Chloro-2,4-dinitrobenzene (CDNB) lowers [reduced glutathione] and increases passive K⁺ transport in human red blood cells (RBCs) (Shartava et al. 2000; Muzyamba et al. 2001). The major passive K⁺ transport pathways are the K⁺ÐCl^– cotransporter (KCC) and the Ca²⁺-activated K⁺ channel (Gardos channel, I_K), but there is some discrepancy about which system represents the main target of CDNB.

Normal human RBCs (HbA cells) and RBCs from sickle cell patients (HbS cells), taken with ethical permission from consenting volunteers, were pretreated for 60 min at 10 % haematocrit (Hct) with or without CDBN (1 mM). They were then equilibrated in air or N₂ prior to measurement of transporter activity at 4 % Hct using ⁸⁶Rb⁺ as a K⁺ congener, in the presence of ouabain (0.1 mM) and bumetanide (1 mM) to obviate transport via Na⁺/K⁺ pump and Na⁺ÐK⁺ÐCl^– cotransporter. KCC activity was determined as the Cl^–-dependent component of K⁺ influx (Cl^– replaced with NO₃^–); I_K as the clotrimazole (5 mM)-sensitive component. ⁴⁵Ca²⁺ was used to measure maximal activity of the plasma membrane Ca²⁺ pump (PCMA) following the methodology of Tiffert et al. (1993). Data are given as means ± S.E.M. (n = 3), fluxes as mmol K⁺ or Ca²⁺ (l cells h)^-1.

In oxygenated HbA cells (K⁺ 5 mM, Ca²⁺ 2.5 mM), KCC activity increased from 0.24 ± 0.04 in controls to 13.22 ± 2.67 with CDNB, and I_K activity from 0.20 ± 0.14 to 5.82 ± 1.54; in N₂, activities in CDNB-treated cells were 3.12 ± 1.65 for KCC and 1.11 ± 0.56 for I_K. Similar effects were observed in oxygenated HbS cells, but there was no inhibition on deoxygenation. In oxygenated CDNB-treated cells, removal of extracellular Ca²⁺ (Ca²⁺-free saline plus 50 mM EGTA) inhibited the activity of KCC by 93 ± 1 %, and there was no observable I_K activity. The protein phosphatase inhibitor, calyculin A (100 nM), inhibited KCC activity by 93 ± 5 % when added prior to CDNB, but had no effect when added afterwards. I_K activity was unaffected by calyculin A. [ATP] and Na⁺/K⁺ pump activity were unaffected by CDNB. Finally, PMCA was reduced by 33 ± 1 % on exposure to CDNB.

The results demonstrate that CDNB stimulates both KCC and I_K. Stimulation is dependent on extracellular Ca²⁺. Inhibition of PCMA may contribute to activation of I_K. Stimulation of KCC is consistent with inhibition of a regulatory protein kinase, but there is no evidence of depletion of total or membrane-bound pools of ATP.

This work was supported by Action Research and The Wellcome Trust.

All procedures accord with current local guidelines and the Declaration of Helsinki.