Characterization of the endogenous Na (+) -K (+) -2Cl- cotransporter in Xenopus oocytes
Suvitayavat, W.; Palfrey, H.C.; Haas, M.; Dunham, P.B.; Kalmar, F.; Rao, M.C.
American Journal of Physiology 266(1 Pt 1): C284-C292
1994
ISSN/ISBN: 0002-9513 PMID: 8304423 Document Number: 429716
Over time, Xenopus laevis changed from producing stage V and VI oocytes with little native Na+-K+-2Cl- cotransport activity to those with substantial activity. In oocytes with high endogenous activity, K+ uptake, using the tracer 86Rb+ was apprx 20 pmol cntdot min-1 cntdot oocyte-1 in the presence of blockers of Na+-K+-ATPase and conductive K+ transport. Bumetanide (10 mu-M) inhibited gt 90% of this uptake, suggesting involvement of Na+-K+-2Cl- cotransport This was confirmed by two observations that are found in this cotransporter in other tissues: 1) The related diuretics, thiobenzmetanide (50% inhibitory concentration (IC-50), 2 times 10-11 M) gt bumetanide (IC-50, 7 times 10-8 M) gt furosemide (IC-50. 2.5 times 10-6 M) inhibited the cotransporter in a dose-dependent manner. 2) There was little uptake of K+ in the absence of extracellular Na+ or Cl-. Halving medium osmolarity to 92 mosM decreased bumetanide-sensitive K+ uptake by apprx 75%, whereas a doubling of medium osmolarity increased it by apprx 50%. The cotransport activity was increased fourfold by the phosphatase inhibitor calyculin A (200 nM) but was unaffected by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. 8-bromoguanosine 3',5'-cyclic monophosphate, ATP, ionomycin, or okadaic acid. Both the photoaffinity bumetanide analogue, 4-(3H)benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid, and an antiserum raised against Ehrlich ascites cell cotransporter specifically labeled an apprx 140-kDa oocyte membrane protein. These results demonstrate that, in addition to the Na+ pump and K+ channels, K+ uptake in Xenopus oocytes occurs via a loop-diuretic-sensitive Na+-K+-2Cl- cotransporter. The putative cotransporter is an apprx 140-kDa membrane protein, and its activity is sensitive to cell volume and changes in protein phosphorylation.