Center for Membrane Pumps in Cells and Disease kin

Århus, Denmark

Center for Membrane Pumps in Cells and Disease kin

Århus, Denmark
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Weigand K.M.,Radboud University Nijmegen | Laursen M.,Center for Membrane Pumps in Cells and Disease kin | Laursen M.,University of Aarhus | Swarts H.G.P.,Radboud University Nijmegen | And 9 more authors.
Chemical Research in Toxicology | Year: 2014

Digitalis-like compounds (DLCs) comprise a diverse group of molecules characterized by a cis-trans-cis ring-fused steroid core linked to a lactone. They have been used in the treatment of different medical problems including heart failure, where their inotropic effect on heart muscle is attributed to potent Na+,K+-ATPase inhibition. Their application as drugs, however, has declined in recent past years due to their small safety margin. Since human Na+,K+-ATPase is represented by four different isoforms expressed in a tissue-specific manner, one of the possibilities to improve the therapeutic index of DLCs is to exploit and amend their isoform selectivity. Here, we aimed to reveal the determinants of selectivity of the ubiquitously expressed α1 isoform and the more restricted α2 isoform toward several well-known DLCs and their hydrogenated forms. Using baculovirus to express various mutants of the α2 isoform, we were able to link residues Met119 and Ser124 to differences in affinity between the α1 and α2 isoforms to ouabain, dihydro-ouabain, digoxin, and dihydro-digoxin. We speculate that the interactions between these amino acids and DLCs affect the initial binding of these DLCs. Also, we observed isoform selectivity for DLCs containing no sugar groups. (Chemical Equation Presented). © 2014 American Chemical Society.


Laursen M.,Center for Membrane Pumps in Cells and Disease kin | Laursen M.,University of Aarhus | Yatime L.,Center for Membrane Pumps in Cells and Disease kin | Yatime L.,University of Aarhus | And 4 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

The Na+,K+-ATPase maintains electrochemical gradients for Na+and K+ that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na+,K+-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na+,K +-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg2+, and crystallographic studies indicate that Rb +and Mn2+, but not Na+, bind to this site. Comparison with the low-affinity [K2]E2-MgFx-ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742-13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg2+. K+binding at the same site unwinds a turn of αM4, dragging residues Ile318-Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K+-Mg2+competition and explain the well-known antagonistic effect of K+on CTS binding.


Winther A.-M.L.,Copenhagen University | Winther A.-M.L.,Center for Membrane Pumps in Cells and Disease kin | Winther A.-M.L.,University of Aarhus | Bublitz M.,Center for Membrane Pumps in Cells and Disease kin | And 9 more authors.
Nature | Year: 2013

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca2+, initiated by the release of Ca2+ from the sarcoplasmic reticulum and terminated by re-sequestration of Ca2+ into the sarcoplasmic reticulum as the main mechanism of Ca2+ removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca2+-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca2+ enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca2+-ATPase in a previously undescribed E1 state, with exposure of the Ca2+ sites through an open cytoplasmic pathway stabilized by Mg2+. The structure suggests a mechanism for selective Ca2+ loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca2+. These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes. © 2013 Macmillan Publishers Limited. All rights reserved.


Nyblom M.,Center for Membrane Pumps in Cells and Disease kin | Nyblom M.,University of Aarhus | Nyblom M.,Novo Nordisk AS | Poulsen H.,Center for Membrane Pumps in Cells and Disease kin | And 16 more authors.
Science | Year: 2013

The Na+, K+-adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na+ and K+ across the plasma membrane - a prerequisite for electrical excitability and secondary transport. Hitherto, structural information has been limited to K +-bound or ouabain-blocked forms. We present the crystal structure of a Na+-bound Na+, K+-ATPase as determined at 4.3 Å resolution. Compared with the K+-bound form, large conformational changes are observed in the α subunit whereas the β and γ subunit structures are maintained. The locations of the three Na+ sites are indicated with the unique site III at the recently suggested IIIb, as further supported by electrophysiological studies on leak currents. Extracellular release of the third Na+ from IIIb through IIIa, followed by exchange of Na+ for K+ at sites I and II, is suggested.


Huliciak M.,Palacky University | Huliciak M.,University of Aarhus | Huliciak M.,Center for Membrane Pumps in Cells and Disease kin | Reinhard L.,University of Aarhus | And 10 more authors.
Biochemical Pharmacology | Year: 2014

Cisplatin is the most widely used chemotherapeutics for cancer treatment, however, its administration is connected to inevitable adverse effects. Previous studies suggested that cisplatin is able to inhibit Na+/K+-ATPase (NKA), the enzyme responsible for maintaining electrochemical potential and sodium gradient across the plasma membrane. Here we report a crystallographic analysis of cisplatin bound to NKA in the ouabain bound E2P form. Despite a moderate resolution (7.4 A˚ and 7.9 A˚), the anomalous scattering from platinum and a model representation from a recently published structure enabled localization of seven cisplatin binding sites by anomalous difference Fourier maps. Comparison with NKA structures in the E1P conformation suggested two possible inhibitory mechanisms for cisplatin. Binding to Met151 can block the N-terminal pathway for transported cations, while binding to Met171 can hinder the interaction of cytoplasmic domains during the catalytic cycle. © 2014 Elsevier Inc. All rights reserved.


PubMed | University of Aarhus and Center for Membrane Pumps in Cells and Disease kin
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2015

Nanodiscs are disc-shaped self-assembled lipid bilayers encircled by membrane scaffolding proteins derived from Apolipoprotein A-1 (apo A-1). They constitute a versatile tool for studying membrane proteins since reconstitution into nanodiscs allows studies of the membrane proteins in detergent-free aqueous solutions in a lipid bilayer. Here, we apply the technique to the Na(+),K(+)-ATPase (NKA) from pig kidney using Membrane Scaffolding Protein 1 D1 (MSP1D1). Contrary to other reports, the nanodiscs obtained by our protocol are built up of the native lipids originally present in the detergent solubilized sample together with the NKA.

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