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

Blood Cells Mol Dis. 2013 Jun;51(1):9-16. doi: 10.1016/j.bcmd.2013.02.004. Epub 2013 Mar 6.

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) are 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 pH6.0 but not significantly altered at pH8.0, and is abolished at 0°C. Although the molecular identity of this little-studied K(+) efflux pathway of mouse erythrocytes remains unknown, its potential role in the pathophysiology of sickle red cell dehydration will be important for the extrapolation of studies in mouse models of sickle cell disease to our understanding of humans with sickle cell anemia.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amiloride / pharmacology
  • Animals
  • Chlorides / metabolism*
  • Chloroquine / pharmacology
  • Drug Resistance / genetics
  • Erythrocytes / drug effects*
  • Erythrocytes / metabolism*
  • Ethylmaleimide / pharmacology*
  • Hemoglobin, Sickle / metabolism
  • Intermediate-Conductance Calcium-Activated Potassium Channels / genetics
  • Intermediate-Conductance Calcium-Activated Potassium Channels / metabolism
  • K Cl- Cotransporters
  • Mice
  • Mice, Knockout
  • Potassium / metabolism*
  • Symporters / agonists*
  • Symporters / antagonists & inhibitors
  • Symporters / metabolism*
  • Temperature

Substances

  • Chlorides
  • Hemoglobin, Sickle
  • Intermediate-Conductance Calcium-Activated Potassium Channels
  • Kcnn4 protein, mouse
  • Symporters
  • hemoglobin SAD
  • Amiloride
  • Chloroquine
  • Ethylmaleimide
  • Potassium