Oxygen‐dependent K+ fluxes in sheep red cells

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Abstract
This study was designed to investigate the O2 dependence of K+ influx in sheep red cells. Influx was determined using 86Rb+ as a tracer for K+; glass tonometers coupled to a gas mixing pump were used to equilibrate cell samples to the requisite oxygen tension (PO2). Both volume‐ and H+‐stimulated K+ influxes in low potassium‐containing (LK) sheep red cells were approximately doubled on equilibration with O2 relative to influxes measured in N2. O2‐dependent influxes were abolished when Cl− was replaced with NO3−, consistent with mediation by the KCl cotransporter. At pH 7, PO2 required for half‐maximal stimulation was 56 ± 1 mmHg (mean ± s.e.m., 3 sheep) for the O2‐dependent component of K+ influx: thus PO2 values over the physiological range affected K+ influx. K+ influx in fully deoxygenated sheep red cells showed substantial volume and H+ sensitivity. These residual components in N2 were also Cl− dependent, indicating that the KCl cotransporter of LK sheep red cells was active in the absence of O2. Volume‐sensitive K+ influxes in high potassium‐containing (HK) sheep red cells responded in a similar way to those in cells from LK sheep, although much smaller in magnitude, showing that intracellular [K+] had no significant effect on the O2 dependence of the cotransporter. Intracellular [Mg2+] ([Mg2+]i) was altered by incubating sheep red cells with A23187 (20 μM) and different values of extracellular [Mg2+] ([Mg2+]o). Total [Mg2+]i was determined by atomic absorption spectroscopy and free [Mg2+]i from [Mg2+]o and the Donnan ratio. Total [Mg2+]i was 1.29 ± 0.08 mM (mean ± s.e.m., n= 5), similar to that reported in the literature. Estimates of free [Mg2+]i showed an increase from 0.39 ± 0.05 in oxygenated cells to 0.52 ± 0.04 mM (mean ± s.e.m., n= 5; P < 0.05) in deoxygenated ones. Finally, although K+ influxes were altered by pharmacological loading or depletion of cells with Mg2+, the free [Mg2+]i required to affect influxes significantly was outside the physiological range. Results are difficult to reconcile with PO2 modulating KCl cotransport activity directly via changes in free [Mg2+]i or [Mg2+‐ATP]i.