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Ouabain affinity determining residues lie close to the Na/K pump ion pathway.

ABSTRACT: The Na/K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na/K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na/K pump and the site of its interaction with ouabain. Palytoxin uncouples the pump's gates, which normally open strictly alternately, thus allowing both gates to sometimes be open, so transforming the pump into an ion channel. Palytoxin therefore permits electrophysiological analysis of even a single Na/K pump. We used outside-out patch recording of Xenopus alpha1beta3 Na/K pumps, which were made ouabain-resistant by point mutation, after expressing them in Xenopus oocytes. Endogenous, ouabain-sensitive, Xenopus alpha1beta3 Na/K pumps were silenced by continuous exposure to ouabain. We found that side-chain charge of two residues at either end of the alpha subunit's first extracellular loop, known to make a major contribution to ouabain affinity, strongly influenced conductance of single palytoxin-bound pump-channels by an electrostatic mechanism. The effects were mimicked by modification of cysteines introduced at those two positions with variously charged methanethiosulfonate reagents. The consequences of these modifications demonstrate that both residues lie in a wide vestibule near the mouth of the pump's ion pathway. Bound ouabain protects the site with the strongest influence on conductance from methanethiosulfonate modification, while leaving the site with the weaker influence unprotected. The results suggest a method for mapping the footprint of bound cardiotonic steroid on the extracellular surface of the Na/K pump.

SUBMITTER: Artigas P

PROVIDER: S-EPMC1567927 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Ouabain affinity determining residues lie close to the Na/K pump ion pathway.

Artigas Pablo P Gadsby David C DC

Proceedings of the National Academy of Sciences of the United States of America 20060807 33

The Na/K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na/K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na/K pump and the site of its interaction with ouabain. Palytoxin uncouples the pump's gates, which normally open strictly alternately, thus allowing both gates to sometimes be open, so transforming the pump into an ion channel ...[more]

PMID: 16894161

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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.

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.

Dataset Information

Ouabain affinity determining residues lie close to the Na/K pump ion pathway.

Publications

Ouabain affinity determining residues lie close to the Na/K pump ion pathway.

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