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

R. Daniel Peluffo, Rodolfo M. González-Lebrero, Sergio B. Kaufman, Sandhya Kortagere, Branly Orban, Rolando C. Rossi, Joshua R. Berlin

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


This study examined how the quaternary organic ammonium ion, benzyltriethylamine (BTEA), binds to the Na,K-ATPase to produce membrane potential (VM)-dependent inhibition and tested the prediction that such a VM-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 VM-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 VM 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 VM, equivalence of charge moved during and after step changes in VM, and similar apparent valence. The rate constant (ktot) for Tl+-dependent transient current asymptotically approached a minimum value at positive VM. In contrast, k tot for pNBTEA-dependent transient current was a "U"-shaped function of VM 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.

Original languageEnglish (US)
Pages (from-to)8105-8119
Number of pages15
Issue number34
StatePublished - Sep 1 2009

All Science Journal Classification (ASJC) codes

  • Biochemistry


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