title: 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.
authors.affiliation: School of Biological Sciences, The University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK. michaelb@liv.ac.uk
authors.forename: Michael
authors.initials: M
authors.lastname: Berenbrink
authors.forename: Susanne
authors.initials: S
authors.lastname: Völkel
authors.forename: Pia
authors.initials: P
authors.lastname: Koldkjaer
authors.forename: Norbert
authors.initials: N
authors.lastname: Heisler
authors.forename: Mikko
authors.initials: M
authors.lastname: Nikinmaa
mesh_terms: D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002264:Carboxylic Acids / Q000494:pharmacology; D002347:Carps / Q000097:blood*; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007192:Indenes / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism*; D010313:Partial Pressure; D011188:Potassium / Q000378:metabolism*; D012414:Rubidium Radioisotopes; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D027981:Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism; D000096922:K Cl- Cotransporters
chemical_list: D002034:Bumetanide; D002264:Carboxylic Acids; D006454:Hemoglobins; D007192:Indenes; D010100:Oxygen; D011188:Potassium; D012414:Rubidium Radioisotopes; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D027981:Symporters; D000096922:K Cl- Cotransporters; C059990:((dihydroindenyl)oxy)alkanoic acid; C100003:deoxyhemoglobin