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 (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.
Peluffo
R Daniel
RD
Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07101-1709, USA.
González-Lebrero
Rodolfo M
RM
Kaufman
Sergio B
SB
Kortagere
Sandhya
S
Orban
Branly
B
Rossi
Rolando C
RC
Berlin
Joshua R
JR
D000818:Animals; D001665:Binding Sites; D004285:Dogs; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D008954:Models, Biological; D008958:Models, Molecular; D009574:Nitro Compounds / Q000737:chemistry / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D004791:Enzyme Inhibitors; D009574:Nitro Compounds; D000644:Quaternary Ammonium Compounds; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium