Cation and Cardiac Glycoside Binding Sites of the Na,K‐ATPase<sup>a</sup>

Lingrel, Jerry B.; Argüello, José M.; Huysse, James Van; Kuntzweiler, Theresa A.

doi:10.1111/j.1749-6632.1997.tb52251.x

Cited by 85 publications

(74 citation statements)

References 41 publications

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“…The Na + /K + ATPase pump is a transmembrane protein that transports potassium ions into and sodium ions out of eukaryotic cells against their natural gradients (34). Cardiac glycosides bind to the pump at a regulatory site that is located on the extracellular side on the a-subunit of the enzyme (35). This inhibition results in an increase in intracellular Na + , and a decrease in intracellular K + (24).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

Simpson

Mawji²,

Anyiwe³

et al. 2009

Cancer Research

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Normal epithelial cells undergo apoptosis upon detachment from the extracellular matrix, a process termed ''anoikis.'' However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. Molecules that sensitize resistant cells to anoikis will be useful chemical probes to understand this pathway. To identify novel anoikis sensitizers in anoikis-resistant PPC-1 prostate adenocarcinoma cells, a library of 2,000 off-patent drugs and natural products was screened for their ability to preferentially induce cell death in suspension over adherent culture conditions. This screen identified five members of the family of cardiac glycosides as anoikis sensitizers, including ouabain, peruvoside, digoxin, digitoxin, and strophanthidin. We conducted further studies with ouabain to discern the mechanism of cardiac glycoside-induced anoikis sensitization. Ouabain initiated anoikis through the mitochondrial pathway of caspase activation. In addition, ouabain sensitized cells to anoikis by inhibiting its known target, the Na + /K + ATPase pump, and inducing hypoosmotic stress. Resistance to anoikis permits cancer cells to survive in the circulation and facilitates their metastasis to distant organs, so we tested the effects of Na + /K + ATPase inhibition on distant tumor formation in mouse models. In these mouse models, ouabain inhibited tumor metastases but did not alter the growth of subcutaneous tumors. Thus, we have identified a novel mechanism to sensitize resistant cells to anoikis and decrease tumor metastasis. These results suggest a potential mechanism for the observed clinical reduction in metastasis and relapse in breast cancer patients who have undergone treatments with cardiac glycosides.

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Section: Discussionmentioning

confidence: 99%

Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

Simpson

Mawji²,

Anyiwe³

et al. 2009

Cancer Research

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“…Mutagenesis has identified residues in the outermost segments of TM1, TM2, TM4, TM5, TM6, and TM7, and their connecting loops, that influence apparent affinity of ouabain binding or action (14, 17, 23, 34-37). Although evidence for direct interaction of these residues with cardiotonic steroids is lacking, the consensus interpretation pictures the steroid docking on the Na͞K pump's external surface, interacting with many of the identified amino acids (14,(38)(39)(40). Identification of the conserved Thr atop TM6 (34) and neighboring residues in the TM5-TM6 hairpin loop (35) as key determinants of ouabain apparent affinity led to the suggestion that cardiotonic steroids might act by preventing motion of the TM5-TM6 hairpin loop, thereby impeding ion passage to or from the deeper binding sites between TM4, TM5, and TM6 (35).…”

Section: Discussionmentioning

confidence: 99%

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

Artigas

Gadsby

2006

Proc. Natl. Acad. Sci. U.S.A.

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The Na͞K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na͞K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na͞K 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 Na͞K pump. We used outside-out patch recording of Xenopus ␣1␤3 Na͞K pumps, which were made ouabain-resistant by point mutation, after expressing them in Xenopus oocytes. Endogenous, ouabain-sensitive, Xenopus ␣1␤3 Na͞K pumps were silenced by continuous exposure to ouabain. We found that side-chain charge of two residues at either end of the ␣ subunit's first extracellular loop, known to make a major contribution to ouabain affinity, strongly influenced conductance of single palytoxin-bound pumpchannels 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 Na͞K pump.Na,K-ATPase ͉ palytoxin ͉ single-channel conductance ͉ cysteine modification ͉ electrostatic mechanism

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“…The genes for the Na,KATPase have been cloned, including isoforms expressed in a variety of different tissues [51,59,71,83]. For the ␣-subunit, consisting of about 1,000 residues, a variety of experimental techniques [9,26,44,46,58,82] have demonstrated the existence of 10 transmembrane segments [45], with both N-and C-termini cytoplasmic. Amino acids that are part of ATP-and cardiac-glycoside binding sites or that are related to the Na + and K + occlusion sites have been identified mainly by mutation experiments.…”

Section: Structural Featuresmentioning

confidence: 99%

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

Apell

Karlish

2001

Journal of Membrane Biology

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Abstract.A full understanding of the molecular mechanism of ion transport and energetics of the Na,K-ATPase will require both structural and functional data. During recent years biophysical methods have provided a number of important pieces of information on ion binding and release and the charge transfer process. This allows the formulation of kinetic models of the transport process. Although a breakthrough has not been obtained due to the lack of detailed knowledge on the threedimensional structure with a resolution high enough to identify the ion-binding sites and the transport pathway, the functional information has structural implications that create constraints on possible mechanisms of active transport. Here we describe briefly contributions of some biophysical methods to our conceptual understanding of the ion transport process.

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Cation and Cardiac Glycoside Binding Sites of the Na,K‐ATPase^a

Cited by 85 publications

References 41 publications

Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

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

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

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Cation and Cardiac Glycoside Binding Sites of the Na,K‐ATPasea

Cited by 85 publications

References 41 publications

Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

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

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

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About

Cation and Cardiac Glycoside Binding Sites of the Na,K‐ATPase^a