Two different oxygen sensors regulate oxygen-sensitive K+ transport in crucian carp red blood cells

摘要

The O2 dependence of ouabain-independent K⁺ transport mechanisms has been studied by unidirectional Rb⁺ flux analysis in crucian carp red blood cells (RBCs). The following observations suggest that O2 activates K⁺–Cl⁻ cotransport (KCC) and deactivates Na⁺–K⁺–2Cl⁻ cotransport (NKCC) in these cells via separate O2 sensors that differ in their O2 affinity. When O2 tension (PO2) at physiological pH 7.9 was increased from 0 to 1, 4, 21 or 100 kPa, K⁺ (Rb⁺) influx was increasingly inhibited, and at 100 kPa amounted to about 30% of the value at 0 kPa. This influx was almost completely Cl⁻ dependent at high and low PO2, as shown by substituting Cl⁻ with nitrate or methanesulphonate. K⁺ (Rb⁺) efflux showed a similar PO2 dependence as K⁺ (Rb⁺) influx, but was about 4–5 times higher over the whole PO2 range. The combined net free energy of transmembrane ion gradients favoured net efflux of ions for both KCC and NKCC mechanisms. The KCC inhibitor dihydroindenyloxyalkanoic acid (DIOA, 0.1 mm) abolished Cl⁻-dependent K⁺ (Rb⁺) influx at a PO2 of 100 kPa, but was only partially effective at low PO₂ (0–1 kPa). At P_O2 values between 0 and 4 kPa, K⁺ (Rb⁺) influx was further unaffected by variations in pH between 8.4 and 6.9, whereas the flux at 21 and 100 kPa was strongly reduced by pH values below 8.4. At pH 8.4, where K⁺ (Rb⁺) influx was maximal at high and low P_O2, titration of K⁺ (Rb⁺) influx with the NKCC inhibitor bumetanide (1, 10 and 100 μm) revealed a highly bumetanide-sensitive K⁺ (Rb⁺) flux pathway at low P_O2, and a relative bumetanide-insensitive pathway at high P_O2. The bumetanide-sensitive K⁺ (Rb⁺) influx pathway was activated by decreasing P_O2, with a P_O2 for half-maximal activation (P₅₀) not significantly different from the P₅₀ for haemoglobin O₂ binding. The bumetanide-insensitive K⁺ (Rb⁺) influx pathway was activated by increasing P_O2 with a P₅₀ significantly higher than for haemoglobin O₂ binding. These results are relevant for the pathologically altered O₂ sensitivity of RBC ion transport in certain human haemoglobinopathies.