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NEM-stimulated Cl−-independent K+ efflux of mouse red cells is insensitive to ouabain and bumetanide, but partially inhibited by chloroquine, barium, and amiloride.
This was different from RBUP which agglutinated rabbit red cells instead of mouse red cells.
Deoxygenation-induced currents in SAD mouse red cells were completely reversible.
Human AA red cells and normal mouse red cells exhibited no deoxygenation-induced increases in current or in [Ca2+]i.
The absence of KCa3.1 in otherwise normal mouse red cells had no effect on the lack of deoxygenation-sensitive [Ca2+]i elevation.
Measurement of relative changes in [Ca2+]i by Fluo-3 fluorescence emission was previously validated in intact human [36], [56] and mouse red cells [63].
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The data demonstrate the activation by deoxygenation of nonspecific cation channel activity in the SAD mouse red cell membrane.
SAD mouse red cell membrane patches sustained GΩ seals during transitions from room air to nitrogen, and then back to room air.
As shown in Figure 2B, deoxygenation increased mean NPo in SAD mouse red cell patches from 0.01±0.013 to 0.48±0.20 (n = 6; p<0.05).
The on-cell patch records of Figure 1A, with NaCl in both pipette and bath, show that deoxygenation activates noisy channel activity in the SAD sickle mouse red cell membrane.
Buffy coat-depleted cells were washed 5 times in standard mouse red cell wash solution containing (in mM) 172 choline Cl, 10 sucrose, 10 Na Tris-MOPS, pH 7.4, resuspended in storage solution, and kept at 4°C until use.
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