N-ethylmaleimide activates a Cl−-independent component of K+ flux in mouse erythrocytes

Abstract

The K-Cl cotransporters (KCCs) of mouse erythrocytes exhibit higher basal activity than those of human erythrocytes, but are similarly activated by cell swelling, by hypertonic urea, and by staurosporine. However, the dramatic stimulation of human erythroid KCCs by N-ethylmaleimide (NEM) is obscured in mouse erythrocytes by a prominent NEM-stimulated K+ efflux that lacks Cl−-dependence. The NEM-sensitivity of Cl−-independent K+ efflux of mouse erythrocytes is lower than that of KCC. The genetically engineered absence of the K-Cl cotransporters KCC3 and KCC1 from mouse erythrocytes does not modify Cl−-independent K+ efflux. Mouse erythrocytes genetically devoid of the Gardos channel KCNN4 show increased NEM-sensitivity of both Cl−-independent K+ efflux and K-Cl cotransport. The increased NEM-sensitivity and stimulation magnitude of Cl−-independent K+ efflux in mouse erythrocytes expressing transgenic hypersickling human hemoglobin SAD (HbSAD) is independent of the presence of KCC3 and KCC1, but absence of KCNN4 reduces the stimulatory effect of HbSAD. NEM-stimulated Cl−-independent K+ efflux of mouse red cells is insensitive to ouabain and bumetanide, but partially inhibited by chloroquine, barium, and amiloride. The NEM-stimulated activity is modestly reduced at pH 6.0, but not significantly altered at pH 8.0, and abolished at 0°C. Although the molecular identity of this little-studied K+ efflux pathway of mouse erythrocytes remains unknown, it’s potential role in the pathophysiology of sickle red cell dehydration will be important for extrapolation of studies in mouse models of sickle cell disease to our understanding of humans with sickle cell anemia.

The Clinically Tested Gardos Channel Inhibitor Senicapoc Exhibits Antimalarial Activity

“…To test whether senicapoc targets the Gardos channel in malaria-infected erythrocytes, we assessed the ability of P. yoelii to grow in the presence and absence of the channel in vivo using a Gardos knockout (IK-1 Ϫ/Ϫ ) mouse model (10,11). Unexpectedly, accelerated parasite growth was evident in IK-1 Ϫ/Ϫ mice by postinoculation day 7 (3.6% versus 2.2% in IK-1 ϩ/ϩ mice; P ϭ 0.03) (Fig.…”