Ionic effects on bumetanide binding to the activated Na/K/2Cl cotransporter: selectivity and kinetic properties of ion binding sites.

The loop diuretic bumetanide binds specifically to the Na/K/2Cl cotransporter of many cell types including duck erythrocytes. Membranes isolated from these erythrocytes retain the ability to bind bumetanide when cells are exposed to cotransport activity stimuli prior to membrane isolation. An extensive study of the effects of ions on specific [3H]bumetanide binding to such membranes is presented here and compared to the activity of these ions in supporting transport function in intact cells. Both Na+ and K+ enhanced bumetanide binding in a saturable manner consistent with a single-site interaction. The K m for each ion was dependent on the concentration of the other cation suggesting heterotropic cooperative interactions between the Na+ and K+ binding sites. Na+ and K+ were partially replaceable, with the selectivity of the Na+ site being Na+ > Li+ > NH 4 + ; N-methyl-d-glucamine+, choline+ and tetramethylammonium+ also supported a small amount of specific binding when substituted for Na+. The selectivity of the K+ site was K+ ≈ Rb+ > NH 4 + > Cs+; N-methyl-d-glucamine+, choline+ and tetramethylammonium+ were inactive at this site. The results of transport experiments revealed a slightly different pattern. Li+ could partially substitute for Na+ in supporting coteansport, but other monovalent cations were completely inactive. The order of potency at the K+ site was NH 4 + > K+ ≈ Rb+ > Cs+ ≫ other monovalent cations. The effect of Cl- on bumetanide binding was biphasic, being stimulatory at low [Cl-] but inhibitory at high [Cl-]. As this implies the existence of two Cl- binding sites (termed Cl H and Cl L for the “high-” and “low-” affinity sites, respectively) each phase was examined individually. Cl- binding to Cl H could be described by a rectangular hyperbola with a K m of 2.5 mm, while kinetic analysis of the inhibition of bumetanide binding at high [Cl-] revealed that it was of a noncompetitive type (K i = 112.9 mm). The selectivity of anion binding to the two sites was distinct. Cl H was highly selective with Cl- > SCN- > Br-; F-, NO 3 - , ClO 4 - , MeSO 4 - , gluconate- and SO 4 2- were inactive. The efficacy of anion inhibition of binding to Cl L was ClO 4 - > I- > SCN- > NO3 > Cl-; F-, MeSO 4 - , gluconate-, and SO 4 2- were inactive. Thus, Cl H is much more selective than Cl L and largely accounts for the specificity of the system with respect to anion transport. SO 4 - , NO 3 - , I-, SCN- and ClO 4 - did not support cotransport when bound to Cl L and the latter three anions were inhibitory. Mg2+ was found to stimulate binding at a narrowly defined peak around 1.5 mm, but was inhibitory at higher concentrations. Other divalent cations caused a similar inhibition of bumetanide binding but did not exert a stimulatory effect at 1.5 mm. Divalent cations have little effect on cotransport in intact cells at concentrations up to 20 mm, suggesting that their effects on diuretic binding reflect interactions at internally disposed sites. Bumetanide binding was optimal at a pH of 7.8–8.1 and declined sharply as the pH was lowered towards 6. The titration curve correlated well with the effect of pH on cotransport in intact cells; the inhibitory effect of low pH suggests that protonation of the cotransporter may inhibit its function.