Membrane potential-dependent inhibition of the Na ⁺,K ⁺-ATPase by para-nitrobenzyltriethylammonium bromide

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.