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Quaternary benzyltriethylammonium ion binding to the Na,K-ATPase: a tool to investigate extracellular K+ binding reactions
Journal article   Open access   Peer reviewed

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 and Joshua R Berlin
Biochemistry (Easton), v 48(34), pp 8105-8119
01 Sep 2009
DOI: https://doi.org/10.1021/bi900687u
PMID: 19621894
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1021/bi900687uView
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Abstract

Potassium - metabolism Sodium-Potassium-Exchanging ATPase - chemistry Extracellular Space - drug effects Nitro Compounds - chemistry Electric Conductivity Extracellular Space - metabolism Time Factors Nitro Compounds - pharmacology Rubidium - metabolism Binding Sites Rabbits Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors Enzyme Inhibitors - metabolism Enzyme Inhibitors - pharmacology Models, Molecular Rats Sodium-Potassium-Exchanging ATPase - metabolism Animals Membrane Potentials Models, Biological Dogs Quaternary Ammonium Compounds - metabolism Protein Binding Protein Conformation Quaternary Ammonium Compounds - pharmacology
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 information about electrogenic ion binding reactions of the Na,K-ATPase that are not directly measurable during ion transport by this enzyme.

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Biochemistry & Molecular Biology
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