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 Km 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+ greater than Li+ greater than NH4+; 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+ approximately Rb+ greater than NH4+ greater than 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 cotransport, but other monovalent cations were completely inactive. The order of potency at the K+ site was NH4+ greater than K+ approximately Rb+ greater than Cs+ much greater than 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 ClH and ClL for the "high-" and "low-" affinity sites, respectively) each phase was examined individually. Cl- binding to ClH could be described by a rectangular hyperbola with a Km of 2.5 mM, while kinetic analysis of the inhibition of bumetanide binding at high [Cl-] revealed that it was of a noncompetitive type (Ki = 112.9 mM). The selectivity of anion binding to the two sites was distinct. ClH was highly selective with Cl- greater than SCN- greater than Br-; F-, NO3-, ClO4-, MeSO4-, gluconate- and SO4(2-) were inactive. The efficacy of anion inhibition of binding to ClL was ClO4- greater than I- greater than SCN- greater than NO3- greater than Cl-; F-, MeSO4-, gluconate-, and SO4(2-) were inactive. Thus, ClH is much more selective than ClL and largely accounts for the specificity of the system with respect to anion transport.(ABSTRACT TRUNCATED AT 250 WORDS)