Search

We examined the properties of Na + /K + /2Cl − cotransport in cultured mouse mTAL cells with respect to its kinetics, the contribution of K/K exchange to K fluxes mediated by the cotransporter, and [ 3 H]bumetanide binding and turnover numbers in media with varying osmolality. The addition of bumetanide, the replacement of external Na + or the replacement of external Cl − resulted in an almost identical (approx. 50%) decrease in K + influx, suggesting that Na + -dependent, Cl − -dependent, BS K + influx was a measure of Na + /K + /2Cl − cotransport. The kinetics of the BS K + influx revealed a high affinity for external Na + ( apparent K m 7 mM ) and external K + ( apparent K m 1.3 mM ), but a very low affinity for external Cl − apparent K m 67 mM with a two-site model ). Of interest was the finding that none of the K + ( 86 RB + ) efflux was sensitive to bumetanide, suggesting the absence of cotransport mediated K/K exchange in this cell type. Specific [ 3 H]bumetanide binding was a saturable function of free bumetanide concentration with a K d of 0.20 μ M and maximum binding ( B max ) of 0.63 pmol/mg, or about 53 000 sites per cell. Simultaneous transport and bumetanide binding assays yielded a turnover number of 255 min −1 . The omission of external Na + , K + or Cl − reduced specific [ 3 H]bumetanide binding to values indistinguishable from zero. Changing medium osmolarity resulted in a co-ordinate change in BS K + influx and bumetanide binding, with a monotonic increase in both transport and bumetanide binding with increase in osmolality from 200 to 400 mosmol/kg. About 85% of the cotransporter sites were located on the apical side, as in the intact mTAL tubule. The simultaneous measurement of BS ion transport and [ 3 H]bumetanide binding in the mTAL cell may provide valuable insights into the regulation of Na + / K + /2Cl − cotransport in this nephron segment.