Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter

The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site with KD = 3.2 +/- 0.3 microM (n = 9) at 100 mM sodium, 100 mM potassium and 5 mM chloride. When KCl-dependent 22Na transport and tracer [3H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that the K0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner. J.N. George and B.J. Baum, J. Membrane Biol. 94:143-152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both [Na] and [K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 1:1 and K0.5 values of approximately 33 mM for sodium and 23 mM for potassium. In contrast, the dependence on [Cl] is biphasic, with bumetanide binding increasing from 0 to 5 mM chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and 22Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site.