Na+/K+/2Cl- cotransport in medullary thick ascending limb cells: kinetics and bumetanide binding

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 [3H]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 Km 7 mM) and external K+ (apparent Km 1.3 mM), but a very low affinity for external Cl- (apparent Km 67 mM with a two-site model). Of interest was the finding that none of the K+ (86Rb+) efflux was sensitive to bumetanide, suggesting the absence of cotransport mediated K/K exchange in this cell type. Specific [3H]bumetanide binding was a saturable function of free bumetanide concentration with a Kd of 0.20 microM and maximum binding (Bmax) 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 [3H]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 [3H]bumetanide binding in the mTAL cell may provide valuable insights into the regulation of Na+/K+/2Cl- cotransport in this nephron segment.