Membrane Potential-Dependent Inhibition of the Na+,K+-ATPase by para-Nitrobenzyltriethylammonium Bromide

Membrane potential (VM)-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 VM-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 VM-dependent Na+,K+-ATPase inhibitor, para-nitrobenzyltriethylamine (pNBTEA), binds to the enzyme by measuring the extracellular pNBTEA concentration and VM 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 VM-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 VM-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 VM-dependent Na+,K+-ATPase inhibitors should be feasible to design and test as specific enzyme inhibitors.