Elucidation of the Na+, K+-ATPase digitalis binding site

Keenan, Susan M.; DeLisle, Robert Kirk; Welsh, William J.; Paula, Stefan; Ball, William J.

doi:10.1016/j.jmgm.2005.02.001

Cited by 40 publications

(30 citation statements)

References 49 publications

(65 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…7. Indeed, two recent models posit that ouabain docks in that cavity, albeit with very different orientations (39,40). Our observation that bound ouabain prevents access of MTS reagent to a Cys substituted at position 131 ( Fig.…”

Section: Discussionmentioning

confidence: 59%

“…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%

“…5B, 6B, and 8). This finding provides the basis for a method of mapping the footprint of a bound steroid, and, hence, of distinguishing between docking models (39,40), by evaluating the steroid's ability to protect introduced Cys from modification by hydrophilic suflhydryl reagents, such as MTSET ϩ .…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

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

Artigas

Gadsby

2006

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

View full text Add to dashboard Cite

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

show abstract

Section: Discussionmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Artigas

Gadsby

2006

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

View full text Add to dashboard Cite

show abstract

“…Consequently, xenobiotic-mediated interference with its role in homeostasis can have catastrophic effects manifesting in cardiotoxicity. For example, interference with ion homeostasis by channel (1) or exchanger blockade (2), altered coronary blood flow, oxidative stress, organellar dysfunction, and apoptosis are all potential mechanisms of cardiotoxicity (3). Two different proteins, namely, the 5-HT 2B receptor and the human ether-a-go-go-related gene (hERG) 1 potassium channel, have raised particular concern primarily due to their association with cardiac valve disease or potassium channel blockade, respectively.…”

Section: Introductionmentioning

confidence: 99%

Shape Signatures: New Descriptors for Predicting Cardiotoxicity In Silico

Chekmarev

Kholodovych

Balakin

et al. 2008

Chem. Res. Toxicol.

Self Cite

View full text Add to dashboard Cite

Shape Signatures is a new computational tool that is being evaluated for applications in computational toxicology and drug discovery. The method employs a customized ray-tracing algorithm to explore the volume enclosed by the surface of a molecule and then uses the output to construct compact histograms (i.e., signatures) that encode for molecular shape and polarity. In the present study, we extend the application of the Shape Signatures methodology to the domain of computational models for cardiotoxicity. The Shape Signatures method is used to generate molecular descriptors that are then utilized with widely used classification techniques such as k nearest neighbors (k-NN), support vector machines (SVM), and Kohonen self-organizing maps (SOM). The performances of these approaches were assessed by applying them to a data set of compounds with varying affinity toward the 5-HT 2B receptor as well as a set of human ether-a-go-go-related gene (hERG) potassium channel inhibitors. Our classification models for 5-HT 2B represented the first attempt at global computational models for this receptor and exhibited average accuracies in the range of 73−83%. This level of performance is comparable to using commercially available molecular descriptors. The overall accuracy of the hERG Shape Signatures-SVM models was 69−73%, in line with other computational models published to date. Our data indicate that Shape Signatures descriptors can be used with SVM and Kohonen SOM and perform better in classification problems related to the analysis of highly clustered and heterogeneous property spaces. Such models may have utility for predicting the potential for cardiotoxicity in drug discovery mediated by the 5-HT 2B receptor and hERG.

show abstract

“…In our opinion, both docking models are plausible. Keenan et al [28] have postulated a model in which the lactone ring is located close to the extracellular space and the rhamnose is embedded deeply into the membrane. The latter orientation does not fit with earlier nuclear magnetic resonance studies showing that the rhamnose has a high mobility [29].…”

Section: Discussionmentioning

confidence: 99%