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 † This research was supported by the National Institutes of Health (R01 HL-076392 to RDP; R01 GM-057253 to JRB),