title: Membrane potential-dependent inhibition of the Na+,K+-ATPase by para-nitrobenzyltriethylammonium bromide.
abstract: Membrane potential (V(M))-dependent inhibitors of the Na(+),K(+)-ATPase are a new class of compounds that may have inherent advantages over currently available drugs targeting this enzyme. However, two questions remain unanswered regarding these inhibitors: (1) what is the mechanism of V(M)-dependent Na(+),K(+)-ATPase inhibition, and (2) is their binding affinity high enough to consider them as possible lead compounds? To address these questions, we investigated how a recently synthesized V(M)-dependent Na(+),K(+)-ATPase inhibitor, para-nitrobenzyltriethylamine (pNBTEA), binds to the enzyme by measuring the extracellular pNBTEA concentration and V(M) dependence of ouabain-sensitive transient charge movements in whole-cell patch-clamped rat cardiac ventricular myocytes. By analyzing the kinetics of charge movements and the steady-state distribution of charge, we show that the V(M)-dependent properties of pNBTEA binding differ from those for extracellular Na(+) and K(+) binding, even though inhibitor binding is competitive with extracellular K(+). The data were also fit to specific models for pNBTEA binding to show that pNBTEA binding is a rate-limiting V(M)-dependent reaction that, in light of homology models for the Na(+),K(+)-ATPase, we interpret as a transfer reaction of pNBTEA from a peripheral binding site in the enzyme to a site near the known K(+) coordination sites buried within the transmembrane helices of the enzyme. These models also suggest that binding occurs with an apparent affinity of 7 μM. This apparent binding affinity suggests that high-affinity V(M)-dependent Na(+),K(+)-ATPase inhibitors should be feasible to design and test as specific enzyme inhibitors.
authors.affiliation: Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07101-1709, USA.
authors.forename: R Daniel
authors.initials: RD
authors.lastname: Peluffo
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Berlin
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001965:Bromides / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D010042:Ouabain / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D051381:Rats; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D001965:Bromides; D004791:Enzyme Inhibitors; D000644:Quaternary Ammonium Compounds; C066585:triethylammonium cation; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium