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

Abstract

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 (EDA) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (I), 2) passive inward H current at negative voltages without Na or K (I), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na (Q). Both amines competed with K to inhibit I. TPA inhibited I without competing with H, whereas EDA did not alter I at pH 7.6. TPA competed with Na in Q 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 (Q). In contrast, EDA and K did not compete with Na to inhibit Q; both reduced Q without decreasing Na-apparent affinity. EDA (15 mM) right-shifted the charge-voltage curve by ∼+50 mV. Simultaneous occlusion of EDA and Na by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA-bound pumps are much more proteolysis-resistant than Na-, K-, or TPA-bound pumps, therefore uncovering unique EDA-bound conformations. K effects on Q and I were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K reduced Q without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited I 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 Q and I, suggesting that there are two external access pathways for proton transport by I.

FXYD protein isoforms differentially modulate human Na/K pump function

“…A previous report measuring the effect of cow FXYD2 on sheep α1β1 did not observe a shift of the Q-V curve ( Dempski et al, 2008 ). It is possible that the FXYD2 effect was lost either due to the use of isoform subunits from different species, the left-shifted nature of this α1β1 construct’s Q-V curve compared with others (compare Stanley et al, 2018 ), or significant differences in 6 of 18 intracellular domain residues between cow and human FXYD2.…”

“…An effect on the E1P/E2P equilibrium suffices to explain the changes in apparent affinity for extracellular Na + , because their effects on ion binding and conformational equilibrium are inseparable with the experiments presented here. Future studies will attempt to dissect direct effects on ion binding from effects on the major conformational equilibrium using techniques with faster time resolution or that report conformational changes concomitantly with the electric signals, such as voltage clamp fluorometry ( Stanley et al, 2018 ).…”

Distinct effects of Q925 mutation on intracellular and extracellular Na+ and K+ binding to the Na+, K+-ATPase

“…7) enclose a cavity containing the ion 14 . The bound Na + ion may possess some freedom to move in the cavity, which does not fit the ion tightly, as suggested by the exclusive ability of site III to house a guanidinium ion in place of Na + 21,25–27 . A clue to how much freedom of movement the Na + ion at site III has in the wild type enzyme comes from comparing the location of this Na + ion in the two slightly different conformations of the α-subunit found in the crystal structure (in protomers A and B, see Fig.…”

Displacement of the Na + /K + pump’s transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases

“…However, interpretation of the functional effects of cross-linking can be difficult (26). We used VCF to gain insight into the movement of M1-M2, utilizing a sheep α1 template, which has been previously evaluated by VCF (27)(28)(29)(30). This template has reduced affinity for ouabain thanks to a double substitution Q116R/N127D and extracellular cysteines removed by the double substitution (C911S and C964A).…”