Occlusion of K+ in the Na+/K+/2Cl- cotransporter of Ehrlich ascites tumor cells.
Proteins of n-octyl glucoside solubilized membrane vesicles derived from Ehrlich ascites tumor cells can occlude 86Rb+.K+ displaces 86Rb+ and it is assumed that 86Rb+ can be used as a tracer to measure K+ occlusion. The following observations indicate that the Na+/K+/2Cl- cotransporter is responsible for this occlusion: (1) Na+ does not compete for the K+ binding site, but rather stimulates 86Rb+ occlusion. (2) K+ occlusion saturates with increasing [Na+] and [K+], the respective K0.5 values being 50 +/- 7 microM for Na+ and 371 +/- 63 microM for K+. (3) Preincubation with 1 mM ouabain does not inhibit 86Rb+ occlusion, arguing against the Na+/K+-ATPase as being responsible for the occlusion. This notion is supported by the K0.5 value for K+ being higher than reported for Na+/K+-ATPase and by the stimulatory effect of Na+. (4) The K+ occlusion is sensitive to [Cl-], and the occluded ion is protected by the presence of bumetanide during cation exchange chromatography. Our results suggest that occlusion measurements of substrate ions could be a profitable way to study the ion binding mechanism(s) of the Na+/K+/2Cl- cotransporter.
Krarup
T
T
Biochemical Department, August Krogh Institute, University of Copenhagen, Denmark. tkrarup@aki.ku.dk
Jensen
B S
BS
Hoffmann
E K
EK
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D017136:Ion Transport; D011188:Potassium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D028021:Sodium-Potassium-Chloride Symporters; D011188:Potassium