Murine models of sickle disease

Murine models of sickle disease have been valuable tools in the study of human SCD pathogenesis [15], [16], and will continue to be central to development of novel approaches to disease treatment. The SAD mouse model of sickle disease is particularly remarkable for its erythrocyte dehydration phenotype in concert with elevated K–Cl cotransport [17] and Gardos channel pathways [18]. Thus, characterization of mouse erythrocyte K+ leak pathways and their regulation are important prerequisites for understanding the range of conditions in which erythrocytes from mouse models of sickle disease faithfully model the dehydration pathology of human sickle erythrocytes. The mouse erythroid Ca-activated K+ channel of intermediate conductance, KCNN4 [19], mediates erythroid Gardos channel activity [20]. Mouse erythrocyte K–Cl cotransport activity is mediated largely by KCC3/SLC12A6, with a small contribution from KCC1/SLC12A4 [21]. Mouse erythroid K–Cl cotransport activity is activated by hypotonic swelling, by staurosporine [22], and by Ketoprofen pH (Shmukler, Casula, LeClair, and Alper, unpublished), as is the corresponding human erythroid activity [23], [24]. However, whereas human erythroid K–Cl cotransport activity is strongly activated by N-ethylmaleimide (NEM) [25], mouse erythroid K+ efflux stimulated by 0.5mM NEM was reduced at most 35% by extracellular Cl− substitution [22]. Only the Cl−-dependent component of NEM-stimulated K+ efflux was inhibited by phosphatase inhibitor okadaic acid or by K–Cl cotransport inhibitorDIOA ([(2-n-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). These data suggested the possible presence in mouse erythrocytes of an independent, alkylation-sensitive K+ leak pathway not evident in human erythrocytes. In the current work, we characterize the NEM-stimulated Cl−-independent K+ efflux pathway of mouse erythrocytes. We show that its sensitivity to NEM is lower than that of K–Cl cotransport, but is enhanced in the presence of transgenic HbSAD. Neither KCNN4, KCC3, nor KCC1 were essential for NEM stimulation of erythroid Cl−-independent K+ efflux. NEM-stimulated Cl−-independent K+ efflux was insensitive to activation by low temperature, and was partially blocked by chloroquine, barium, and amiloride, but not by DIDS (4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid) or by other tested cation transport inhibitors.
Materials and Methods
Results
Discussion The ability of nucleated cells to maintain a cytosolic composition of high [K+] and low [Na+] while bathed in extracellular fluid of high [Na+] and low [K+] has long focused interest on the identity and structure of transmembrane proteins that regulate membrane cation permeability. This interest has prompted investigation of the molecular components of the “leak” that contribute to the established pump-leak balance of the erythrocyte. Erythroid membrane cation “leak” has been widely defined as cation transport insensitive to both the inhibitor of Na+,K+-ATPase, ouabain, and to the inhibitor of Na-K-2Cl cotransport, bumetanide. Inhibition of pathways contributing to this ouabain- and bumetanide-insensitive permeability can control volume and intracellular Hb concentration in circulating erythrocytes, with therapeutic potential in SCD as illustrated by clinical trials of clotrimazole [44] and its more specific congener, senicapoc [13]. In the current work, we identify a novel cation leak pathway in mouse erythrocytes that may contribute to the defense of erythrocyte integrity in pro-hemolytic settings. This leak pathway may be of physiological importance in mouse models of sickle disease. A potentially similar pathway of NEM-sensitive Cl−-independent K+ efflux has been noted in human erythrocytes, although requiring NEM concentrations >0.5mM [45]. Peroxynitrite treatment of human erythrocytes also stimulated Cl−-independent K+ flux, in a manner inhibited 75% by deoxygenation and reduced 40% by 0.2 pH units extracellular acidification [46]. In contrast, NEM inhibited Cl−-independent K+ flux in trout erythrocytes [47].