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J Biol Chem
2011 Nov 04;28644:38177-38183. doi: 10.1074/jbc.M111.267682.
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Ouabain binding site in a functioning Na+/K+ ATPase.
Sandtner W
,
Egwolf B
,
Khalili-Araghi F
,
Sánchez-Rodríguez JE
,
Roux B
,
Bezanilla F
,
Holmgren M
.
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The Na(+)/K(+) ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na(+)/K(+) ATPase sequence. These five normally functional LBT-Na(+)/K(+) ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na(+)/K(+) ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na(+)/K(+) ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies.
Adams,
PHENIX: building new software for automated crystallographic structure determination.
2002, Pubmed
Adams,
PHENIX: building new software for automated crystallographic structure determination.
2002,
Pubmed
Antolovic,
Labeling of a cysteine in the cardiotonic glycoside binding site by the steroid derivative HDMA.
1995,
Pubmed
,
Xenbase
Brinkmann,
Fluoresceinyl-ethylenediamine-ouabain detects an acidic environment in the cardiac glycoside binding site of Na+/K+-ATPase.
1997,
Pubmed
Canessa,
Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase alpha subunit confers ouabain resistance.
1992,
Pubmed
,
Xenbase
Canessa,
Mutation of a tyrosine in the H3-H4 ectodomain of Na,K-ATPase alpha subunit confers ouabain resistance.
1993,
Pubmed
,
Xenbase
Colina,
Structural basis of Na(+)/K(+)-ATPase adaptation to marine environments.
2007,
Pubmed
Collaborative Computational Project, Number 4,
The CCP4 suite: programs for protein crystallography.
1994,
Pubmed
Croyle,
Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding.
1997,
Pubmed
de Carvalho Aguiar,
Mutations in the Na+/K+ -ATPase alpha3 gene ATP1A3 are associated with rapid-onset dystonia parkinsonism.
2004,
Pubmed
De Fusco,
Haploinsufficiency of ATP1A2 encoding the Na+/K+ pump alpha2 subunit associated with familial hemiplegic migraine type 2.
2003,
Pubmed
De Pont,
The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase.
2009,
Pubmed
Emsley,
Features and development of Coot.
2010,
Pubmed
Forbush,
Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na, K-ATPase.
1978,
Pubmed
Gadsby,
Extracellular access to the Na,K pump: pathway similar to ion channel.
1993,
Pubmed
Heyduk,
Luminescence energy transfer with lanthanide chelates: interpretation of sensitized acceptor decay amplitudes.
2001,
Pubmed
Hilgemann,
Channel-like function of the Na,K pump probed at microsecond resolution in giant membrane patches.
1994,
Pubmed
Holmgren,
Three distinct and sequential steps in the release of sodium ions by the Na+/K+-ATPase.
2000,
Pubmed
Jaisser,
Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump.
1992,
Pubmed
,
Xenbase
Jeffrey,
Structure and specificity of the anti-digoxin antibody 40-50.
1995,
Pubmed
Kawamura,
Evidence for essential disulfide bonds in the beta-subunit of (Na+ + K+)-ATPase.
1984,
Pubmed
Kirley,
Determination of three disulfide bonds and one free sulfhydryl in the beta subunit of (Na,K)-ATPase.
1989,
Pubmed
Lingrel,
Regulation of the alpha-subunit genes of the Na,K-ATPase and determinants of cardiac glycoside sensitivity.
1991,
Pubmed
Lutsenko,
An essential role for the extracellular domain of the Na,K-ATPase beta-subunit in cation occlusion.
1993,
Pubmed
Martínez-François,
Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore.
2010,
Pubmed
,
Xenbase
Martí-Renom,
Comparative protein structure modeling of genes and genomes.
2000,
Pubmed
Middleton,
Structural insights into the binding of cardiac glycosides to the digitalis receptor revealed by solid-state NMR.
2000,
Pubmed
Miller,
Beta subunit of (Na+ + K+)-ATPase contains three disulfide bonds.
1990,
Pubmed
Morth,
Crystal structure of the sodium-potassium pump.
2007,
Pubmed
Murshudov,
Efficient anisotropic refinement of macromolecular structures using FFT.
1999,
Pubmed
Nitz,
Structural origin of the high affinity of a chemically evolved lanthanide-binding peptide.
2004,
Pubmed
Ogawa,
Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain.
2009,
Pubmed
Price,
Site-directed mutagenesis of a conserved, extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na,K-ATPase.
1989,
Pubmed
Price,
Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit.
1990,
Pubmed
Price,
Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme.
1988,
Pubmed
Qiu,
Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids.
2005,
Pubmed
Reyes,
Ion permeation through the Na+,K+-ATPase.
2006,
Pubmed
,
Xenbase
Sandtner,
In vivo measurement of intramolecular distances using genetically encoded reporters.
2007,
Pubmed
,
Xenbase
Schaftenaar,
Molden: a pre- and post-processing program for molecular and electronic structures.
2000,
Pubmed
Segall,
Alterations in the alpha2 isoform of Na,K-ATPase associated with familial hemiplegic migraine type 2.
2005,
Pubmed
Selvin,
Principles and biophysical applications of lanthanide-based probes.
2002,
Pubmed
Shinoda,
Crystal structure of the sodium-potassium pump at 2.4 A resolution.
2009,
Pubmed
Stürmer,
Fluorescence study on cardiac glycoside binding to the Na,K-pump. Ouabain binding is associated with movement of electrical charge.
1992,
Pubmed
Takeuchi,
The ion pathway through the opened Na(+),K(+)-ATPase pump.
2008,
Pubmed
,
Xenbase
Vanmolkot,
Novel mutations in the Na+, K+-ATPase pump gene ATP1A2 associated with familial hemiplegic migraine and benign familial infantile convulsions.
2003,
Pubmed
Yatime,
Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase.
2011,
Pubmed
Zhou,
Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors.
2001,
Pubmed
,
Xenbase
