Binding of nitrate to renal brush border membranes studied with 14N nuclear magnetic resonance (NMR)

Abstract

Renal brush border membranes contain several anion exchangers that may play a role in electrolyte transport and pH regulation. To help characterize the types of exchangers present and the binding properties of these membranes, the binding of nitrate (NO 3 −) to highly purified rabbit kidney brush border membrane vesicles was studied. The method is based on the binding induced quadrupole relaxation of the 14N-NMR signal of nitrate [1,2]. Brush border membrane vesicles caused a relaxation of the 14N-NMR nitrate signal which could be characterized by relatively high affinity sites, K D = 6.7 ± 1.5 mM, as well as nonspecific interactions with the membranes, K D > 150 mM. The anion transport inhibitor 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS) inhibited 51 ± 6% ( n = 4) of the relaxation due to the high affinity binding sites. The DNDS inhibition could be characterized by a K i of 10–80 μM. Both bicarbonate and formate (HCO 2 −) were found to partially inhibit the high affinity induced relaxation, with maximal inhibition of 37 ± 8% ( n = 3) and 30 ± 2% ( n = 3), respectively. The inhibitory effects of saturating concentrations of bicarbonate and formate were non-additive, suggesting the existence of a stilbene sensitive exchanger that can bind nitrate, as well as both bicarbonate and formate. This study indicates the usefulness of this new method for further investigation of anion exchangers on these and other membranes.