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