K⁺ transport is markedly abnormal in RBCs from sickle cell patients, homozygous for HbS (HbSS) (Ellory et al. 1998). HbS polymerises on deoxygenation, and this is accompanied by the appearance of a non-specific cation channel (P_sickle, Lew et al. 1997). P_sickle supports entry of Ca²⁺, thereby stimulating the Ca²⁺-activated K⁺ channel (Gardos channel, probably I_K1). Unlike in normal (HbA-containing) RBCs, the K⁺-Cl^– cotransporter (probably KCC1) remains active, even in fully deoxygenated sickle cells (Gibson et al. 1998). These three transport processes result in rapid loss of K⁺ and Cl^–, cell shrinkage and elevation of [Hb], thus exacerbating cell sickling. RBCs from patients heterozygous for HbS and HbC (HbSC) also show Hb polymerisation at low O₂ tension; however, the characteristics of their K⁺ transport have not been fully established.

HbSC-containing RBCs, obtained with consent and ethical permission, were suspended in saline containing (mM): 80 KCl, 70 NaCl, 0.15 MgCl₂, 2.5 CaCl₂, 10 inosine and 10 MOPS; pH 7.4 at 37 °C, equilibrated with N₂ or air. ⁸⁶Rb⁺ was used a K⁺ congener to measure KCC1 (Cl^–-dependent K⁺ influx) and I_K1 (clotrimazole, 5 µM, -sensitive K⁺ influx) activity, in cells swollen anisotonically by 10 % at pH 7. Ouabain (0.1 mM) and bumetanide (0.01 mM) were present to inhibit transport via the Na⁺ pump and Na⁺-K⁺-2Cl^– cotransporter. RBC samples were also fixed in saline containing 1 % glutaraldehyde for analysis of cell shape. See Gibson et al. (2001) for full methodology.

In oxygenated HbSC RBCs, I_K1, KCC1 and Cl^–-independent K⁺ influxes were 0, 16.38 ± 2.72 and 6.46 ± 1.03 mmol (l cells h)^-1, respectively (means ± S.E.M., n = 3). In deoxygenated cells, I_K1 activity increased to 8.08 ± 2.91 and Cl^–-independent K⁺ influx to 10.57 ± 1.67; KCC1 activity fell to 0.99 ± 1.51. The percentage of sickled cells was 0 % in O₂ and 88 ± 3 % in N₂.

Thus, as expected, HbSC-containing RBCs sickled when deoxygenated. The rise in Cl^–-independent K⁺ influx indicates opening of P_sickle, and I_K1 was activated. KCC1 activity, however, although higher than for normal RBCs, remained O₂ sensitive (94 ± 10 % inhibition on deoxygenation). The difference in response of KCC1 in HbSS and HbSC RBCs is important for understanding the pathophysiology of sickle cell disease.

We thank The Wellcome Trust and Action Research for financial support.

Ellory, J.C., Gibson, J.S. & Stewart, G.W. (1998). Contrib. Nephrol. 123, 220-239.

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

Gibson, J.S., Khan, A., Speake, P.F. & Ellory, J.C. (2001). FASEB J. 15, 823-832.

Lew, V.L., Ortiz, O.E. & Bookchin, R.M. (1997). J. Clin. Invest. 99, 2727-2735.