External Ion Access in the Na/K Pump: Kinetics of Na+, K+, and Quaternary Amine Interaction

• Published in: Biophysical journal Vol. 115; no. 2; pp. 361 - 374
• Main Authors: Stanley, Kevin S., Young, Victoria C., Gatto, Craig, Artigas, Pablo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.07.2018; The Biophysical Society
• Subjects: Amines - metabolism; Amines - pharmacology; Animals; Channels and Transporters; Ion Transport - drug effects; Kinetics; Models, Molecular; Potassium - metabolism; Protein Binding; Protein Conformation; Sodium - metabolism; Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - metabolism; Xenopus laevis
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2018.06.007

Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na+, with the import of two K+ and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na+, K+, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA+) and ethylenediamine (EDA2+) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (IP), 2) passive inward H+ current at negative voltages without Na+ or K+ (IH), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na+ (QNa). Both amines competed with K+ to inhibit IP. TPA+ inhibited IH without competing with H+, whereas EDA2+ did not alter IH at pH 7.6. TPA+ competed with Na+ in QNa measurements, reducing Na+-apparent affinity, evidenced by a ∼−75 mV shift in the charge-voltage curve (at 20 mM TPA+) without reduction of the total charge moved (Qtot). In contrast, EDA2+ and K+ did not compete with Na+ to inhibit QNa; both reduced Qtot without decreasing Na+-apparent affinity. EDA2+ (15 mM) right-shifted the charge-voltage curve by ∼+50 mV. Simultaneous occlusion of EDA2+ and Na+ by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA2+-bound pumps are much more proteolysis-resistant than Na+-, K+-, or TPA+-bound pumps, therefore uncovering unique EDA2+-bound conformations. K+ effects on QNa and IH were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K+ reduced Qtot without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited IH to the same extent as TPA+ in non-beryllium-fluorinated pumps. These results demonstrate that K+ interacts with beryllium-fluorinated pumps inducing conformational changes that alter QNa and IH, suggesting that there are two external access pathways for proton transport by IH.