Quaternary benzyltriethylammonium ion binding to the Na,K-ATPase: A tool to investigate extracellular K⁺ binding reactions

This study examined how the quaternary organic ammonium ion, benzyltriethylamine (BTEA), binds to the Na,K-ATPase to produce membrane potential (V_M)-dependent inhibition and tested the prediction that such a V_M-dependent inhibitor would display electrogenic binding kinetics. BTEA competitively inhibited K⁺ activation of Na,K-ATPase activity and steady-state ⁸⁶Rb⁺ occlusion. The initial rate of ⁸⁶Rb⁺ occlusion was decreased by BTEA to a similar degree whether it was added to the enzyme prior to or simultaneously with Rb⁺, a demonstration that BTEA inhibits the Na,K-ATPase without being occluded. Several BTEA structural analogues reversibly inhibited Na,K-pump current, but none blocked current in a V_M-dependent manner except BTEA and its para-nitro derivative, pNBTEA. Under conditions that promoted electroneutral K⁺-K⁺ exchange by the Na,K-ATPase, step changes in V_M elicited pNBTEA-activated ouabain-sensitive transient currents that had similarities to those produced with the K⁺ congener, Tl⁺. pNBTEA- and Tl⁺-dependent transient currents both displayed saturation of charge moved at extreme negative and positive V_M, equivalence of charge moved during and after step changes in V_M, and similar apparent valence. The rate constant (k_tot) for Tl⁺-dependent transient current asymptotically approached a minimum value at positive V_M. In contrast, k _tot for pNBTEA-dependent transient current was a "U"-shaped function of V_M with a minimum value near 0 mV. Homology models of the Na,K-ATPase alpha subunit suggested that quaternary amines can bind to two extracellularly accessible sites, one of them located at K⁺ binding sites positioned between transmembrane helices 4, 5, and 6. Altogether, these data revealed important information about electrogenic ion binding reactions of the Na,K-ATPase that are not directly measurable during ion transport by this enzyme.