Dataset Information

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 (EDA²⁺) 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 EDA²⁺ 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, EDA²⁺ and K⁺ did not compete with Na⁺ to inhibit Q_Na; both reduced Q_tot 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 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.

SUBMITTER: Stanley KS

PROVIDER: S-EPMC6051257 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Publications

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

Stanley Kevin S KS Young Victoria C VC Gatto Craig C Artigas Pablo P

Biophysical journal 20180701 2

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- ...[more]

PMID: 30021111

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Project description:Decades ago, it was proposed that Na transport in cardiac myocytes is modulated by large changes in cytoplasmic Na concentration within restricted subsarcolemmal spaces. Here, we probe this hypothesis for Na/K pumps by generating constitutive transsarcolemmal Na flux with the Na channel opener veratridine in whole-cell patch-clamp recordings. Using 25 mM Na in the patch pipette, pump currents decay strongly during continuous activation by extracellular K (?, ?2 s). In contradiction to depletion hypotheses, the decay becomes stronger when pump currents are decreased by hyperpolarization. Na channel currents are nearly unchanged by pump activity in these conditions, and conversely, continuous Na currents up to 0.5 nA in magnitude have negligible effects on pump currents. These outcomes are even more pronounced using 50 mM Li as a cytoplasmic Na congener. Thus, the Na/K pump current decay reflects mostly an inactivation mechanism that immobilizes Na/K pump charge movements, not cytoplasmic Na depletion. When channel currents are increased beyond 1 nA, models with unrestricted subsarcolemmal diffusion accurately predict current decay (? ?15 s) and reversal potential shifts observed for Na, Li, and K currents through Na channels opened by veratridine, as well as for Na, K, Cs, Li, and Cl currents recorded in nystatin-permeabilized myocytes. Ion concentrations in the pipette tip (i.e., access conductance) track without appreciable delay the current changes caused by sarcolemmal ion flux. Importantly, cytoplasmic mixing volumes, calculated from current decay kinetics, increase and decrease as expected with osmolarity changes (? >30 s). Na/K pump current run-down over 20 min reflects a failure of pumps to recover from inactivation. Simulations reveal that pump inactivation coupled with Na-activated recovery enhances the rapidity and effectivity of Na homeostasis in cardiac myocytes. In conclusion, an autoregulatory mechanism enhances cardiac Na/K pump activity when cytoplasmic Na rises and suppresses pump activity when cytoplasmic Na declines.

Project description:The Na(+),K(+)-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na(+) ions for extracellular K(+) ions by using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel. We made a cysteine scan of Na(+),K(+)-ATPase transmembrane (TM) segments TM1 to TM6, and used recordings of Na(+) current flow through palytoxin-bound pump-channels to monitor accessibility of introduced cysteine residues via their reaction with hydrophilic methanethiosulfonate (MTS) reagents. To visualize the open-channel pathway, the reactive positions were mapped onto a homology model of Na(+),K(+)-ATPase based on the structure of the related sarcoplasmicand endoplasmic-reticulum (SERCA) Ca(2+)-ATPase in a BeF(3)(-)-trapped state,(1,2) in which the extra-cytoplasmic gate is wide open (although the cytoplasmic access pathway is firmly shut). The results revealed a single unbroken chain of reactive positions that traverses the pump from the extracellular surface to the cytoplasm, comprises residues from TM1, TM2, TM4 and TM6, and passes through the equivalent of cation binding site II in SERCA, but not through site I. Cavity search analysis of the homology model validated its use for mapping the data by yielding a calculated extra-cytoplasmic pathway surrounded by MTS-reactive residues. As predicted by previous experimental results, that calculated extra-cytoplasmic pathway abruptly broadens above residue T806, at the outermost end of TM6 that forms the floor of the extracellular-facing vestibule. These findings provide a structural basis for further understanding cation translocation by the Na(+),K(+)-ATPase and by other P-type pumps like the Ca(2+)- and H(+),K(+)-ATPases.

Dataset Information

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

Publications

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

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