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

Abstract

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 PO2 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 PO2, titration of K+ (Rb+) influx with the NKCC inhibitor bumetanide (1, 10 and 100 microM) revealed a highly bumetanide-sensitive K+ (Rb+) flux pathway at low PO2, and a relative bumetanide-insensitive pathway at high PO2. The bumetanide-sensitive K+ (Rb+) influx pathway was activated by decreasing PO2, with a PO2 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 PO2 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.

Figure 1. Effect of O₂ tension, DIOA and ion replacements on K⁺ (Rb⁺) fluxes

Cells were pre-equilibrated for 45 min at pH 7.9 and a P_O2 of 1 kPa and then exposed to the indicated O₂ tensions. After 10 min, subsamples were diluted 10-fold in pre-equilibrated salines of the same P_O2 and unidirectional K⁺ (Rb⁺) influx (A and B) and efflux (C) were measured in the presence of 0.1 mm ouabain. A, •, control (n = 8); ○, 0.1 mm dihydroindenyloxyalkanoic acid (DIOA) (n = 4); ▴ and ▵, medium chloride was replaced by nitrate and methanesulphonate, respectively (both n = 3). B, ▪, control; □, sodium salts replaced by NMDG (both n = 3). C, n = 3. All values are means ± s.e.m. *Significantly different from corresponding control value at the same P_O2 (A and B); †significantly different from the corresponding control value and DIOA value at the same P_O2 (A only). Note the logarithmic scale for P_O2.

Figure 2. Net free energy for coupled Na⁺–K⁺–2Cl⁻ and K⁺–Cl⁻ cotransport in crucian carp red blood cells

Lines show the calculated relationships between intracellular Cl⁻ concentration and ΔG for electroneutral cotransport via K⁺–Cl⁻ cotransport (KCC) and Na⁺–K⁺–2Cl⁻ cotransport (NKCC) (stoichiometries of 1:1 and 1:1:2, respectively) in air-equilibrated crucian carp red blood cells. Intracellular Na⁺ and K⁺ concentrations under these conditions were 16 ± 1 and 131 ± 5 mmol (l cell water)⁻¹, respectively (means ± s.e.m., n = 3). Extracellular Na⁺, K⁺ and Cl⁻ concentrations (136, 3 and 135 mm) refer to concentrations in the medium, which was used for diluting RBC suspensions at the start of K⁺ (Rb⁺) flux measurements. Filled circles and error bars indicate values at the measured intracellular Cl⁻ concentration of 68 ± 6 mmol (l cell water)⁻¹ (mean ± s.e.m., n = 3).

Figure 3. Effects of O₂ and pH on K⁺ (Rb⁺) influx and Hb deoxygenation

After standard pre-equilibration (see Fig. 1), RBCs were exposed to P_O2 values between 0 and 100 kPa. After 10 min they were diluted in salines with pH values between 6.9 and 8.4, and these were pre-equilibrated with the same experimental P_O2, and K⁺ (Rb⁺) influx (A) and fractional Hb deoxygenation (C) were measured. For clarity, mean values are shown with s.e.m. in one direction only (circles and bars, respectively). A, two-way analysis of variance with P_O2 and pH as factors revealed a highly significant interaction between the two factors (P≤ 0.001). Thus, the effect of P_O2 on K⁺ (Rb⁺) influx (n = 3–10) depended on pH. ^aSignificantly different from values at 0 kPa at the same pH; ^bsignificantly different from values at pH 8.4 at the same P_O2. B, comparable data on rainbow trout RBCs (n = 3–4), partly taken from Berenbrink et al. (2000). C, fractional deoxyHb in crucian carp RBCs, measured under identical experimental conditions as for K⁺ (Rb⁺) influxes (n = 3). D, reconstruction of the basic shape of P_O2 and pH-dependent K⁺ (Rb⁺) influx as seen in A. At each x, y coordinate, the fraction of deoxyHb from C was added to the fractional K⁺ (Rb⁺) influx taken from B, and the sum is plotted as the z-value. This demonstrates that fluxes in crucian carp RBCs (A) can be modelled in principle by the sum of a deoxyHb-activated flux and a deoxyHb-independent flux, the latter being similar to KCC in rainbow trout RBCs. See text for further details. Note the logarithmic scale for P_O2.

Figure 4. O₂ affinity of K⁺ (Rb⁺) influx and Hb at different pH values

O₂ affinity is expressed as the P_O2 at which half-maximal change in K⁺ (Rb⁺) influxes or Hb O₂ binding occurred (P₅₀, means ± s.e.m). At each pH, P₅₀ values for Hb (filled bars) were calculated by fitting hyperbolic saturation curves to data from three independent experiments (same data as in Fig. 3C). P₅₀ values for K⁺ (Rb⁺) fluxes (n = 5) were calculated by fitting hyperbolic saturation curves (open bars) or hyperbolic decay curves (hatched bars) to the data, as appropriate. These curve fits allowed for a variable, basal O₂-independent K⁺ (Rb⁺) influx component. At pH 6.9, all flux data from Fig. 3A were used. At pH 7.9, only flux data from animals where K⁺ (Rb⁺) influx at P_O2 0 kPa was at least two times higher than at 100 kPa were used (5 out of the 8 independent experiments incorporated into Fig. 3A). At pH 8.4, total K⁺ (Rb⁺) influx could be separated into a 1 μm bumetanide-sensitive component and a 100 μm bumetanide-resistant component (see Fig. 5). n.s., P₅₀ for Hb and total flux did not differ significantly from each other at pH 6.9 and 7.9. At pH 8.4, P₅₀ for bumetanide-sensitive K⁺ (Rb⁺) influx and Hb also did not differ significantly (n.s.). However, both were significantly smaller than the P₅₀ of bumetanide-resistant K⁺ (Rb⁺) influx (**).

Figure 5. O₂ dependence of bumetanide-sensitive and -insensitive K⁺ (Rb⁺) influxes

K⁺ (Rb⁺) influxes (means ± s.e.m., n = 5) were measured as described in Fig. 1, but at pH 8.4 and in the presence of 0, 1, 10 or 100 μm bumetanide, as indicated (A). Two-way analysis of variance with P_O2 and [bumetanide] as factors revealed that there was a highly significant interaction between the two factors (P≤ 0.001). Thus, the effects of bumetanide differed significantly depending on the P_O2 that was present. *Significantly different from the control value in the absence of bumetanide at the same P_O2. †Significantly different from control and from the value in the presence of 1 μm bumetanide at the same P_O2. §Significantly different from the value at P_O2 0 kPa at the same bumetanide concentration. B, 1 μm bumetanide-sensitive K⁺ (Rb⁺) influx, calculated as the difference between fluxes in the absence and presence of 1 μm bumetanide. Mean values not labelled by the same letter differed significantly from each other. Note the logarithmic scale for P_O2.