In RBCs from many species (including humans), K⁺-Cl^– cotransport (KCC) is O₂ dependent (at low P_O2 values, it is inactive, becoming active only at high P_O2 values: Gibson et al. 2000). Free [Mg²⁺]_i in human RBCs falls on oxygenation (from 0.6 to 0.4 mM: Flatman, 1980) and this lower free [Mg²⁺]_i may stimulate KCC, via the regulatory phosphorylation cascade. On the other hand, in RBCs from horse and LK sheep, oxy-deoxy fluctuations in free [Mg²⁺]_i are insufficient to account for O₂ activation of KCC, and KCC activity is stimulated by O₂ in Mg²⁺-clamped cells (see Gibson et al. 2000 for references). In sickle cells, by contrast, KCC is predominantly O₂ independent (Gibson et al. 1998), with activity in Mg²⁺-clamped cells reported to be stimulated by deoxygenation (Joiner et al. 1998). We therefore decided to study the effect of O₂ and Mg²⁺ in normal human RBCs as a necessary prelude to studying sickle cells.

Normal human RBCs (obtained by consent with ethical approval) were Mg²⁺-clamped using A23187 (10 µM) and different [Mg²⁺]_o values, under either oxygenated or deoxygenated conditions, Ca²⁺-free media plus 0.1 mM EGTA to prevent activation of the Gardos channel. KCC activity was determined as Cl^–-dependent component of K⁺ influx (using ⁸⁶Rb as a K⁺ congener; at pH 7, 260 mosmol kg^-1, 37 °C) in the presence of ouabain (0.1 mM) and bumetanide (0.01 mM) to inhibit flux through the Na⁺-K⁺ pump and Na⁺-K⁺-2Cl^– cotransporter. In a typical experiment, KCC activity in oxygenated cells was 1.10 ± 0.02, 0.32 ± 0.03 and 0.20 ± 0.02 mmol (l cells h)^-1 at free [Mg²⁺]_i values of 0.1, 0.3 and 0.6 mM; corresponding values in N₂ were 0.07 ± 0.02, 0.07 ± 0.02 and 0.04 ± 0.02 (means ± S.E.M., n = 3). Estimates of physiological [Mg²⁺]_i, as the free [Mg²⁺]_i in A23187-treated cells giving the same KCC activity as intact RBCs (in the absence of ionophore), were about 0.4 mM for oxygenated cells and 0.6 mM for deoxygenated cells, consistent with Flatman’s measurements. No differences were observed in cell volume or pH_i (data not shown).

We conclude that KCC in Mg²⁺-clamped normal human RBCs remained stimulated by O₂, the opposite response to that seen in sickle cells. Differences in Mg²⁺ sensitivity, via regulatory protein kinases and phosphatases, may account for these observations, elucidation of which is relevant to understanding the pathogenesis of sickle cell disease.

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

Flatman, P.W. (1980). J. Physiol. 300, 19-30. abstract

Gibson, J.S., Cossins, A.R. & Ellory, J.C. (2000). J. Exp. Biol. 203, 1395-1407.

Gibson, J.S., Speake, P.F. & Ellory, J.C. (1998). J. Physiol. 511, 225-234. abstract

Joiner, C.H., Jiang, M., Fathallah, H., Giraud, F. & Franco, R.S. (1998). Am. J. Physiol. 274, C1466-1475.