Quaternary Benzyltriethylammonium Ion Binding to the Na,K-ATPase: A Tool to Investigate Extracellular K + Binding Reactions (original) (raw)

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 86 Rb + occlusion. The initial rate of 86 Rb + 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 . SUPPORTING INFORMATION AVAILABLE Supplemental material contains the following: (1) derivation of a general time-dependent solution for a 3 state model of the Na,K-ATPase in the presence of an activating ligand (K + ) and a competitive inhibitor; (2) a brief review of the V M dependence of Na,K-pump current inhibition by TEA and BTEA and an explanation of how eq. 4 can be used to show V M -dependent inhibition of the Na,K-ATPase, (3) a figure comparing Na,K-pump current in TMA and NMG-containing superfusion solutions; (4) a figure showing the synthetic scheme for para-nitrobenzyltriethylammonium bromide; (5) a figure showing the K + o and V M dependence of Na,K-pump current in the presence and absence of TEA and BTEA, (6) a figure showing transient difference currents observed in the presence of 20 mM TEA; (7) a two part figure of the Na,K-ATPase α subunit homology model with BTEA bound, as viewed from above the extracellular access pathway, and predicted surface charge polarity of the enzyme. This materials is available free of charge via the Internet at