Bente Vilsen Lab

Research

The Na+,K+-ATPase is a membrane bound ion pump that uses energy liberated by hydrolysis of ATP to exchange intracellular Na+ for extracellular K+ at a ratio of 3:2, thereby creating transmembrane gradients for Na+ and K+ of importance for many vital cellular processes including water and salt homeostasis and electrical communication between neurons. The Na+,K+-ATPase consists of a catalytic a-subunit with three cytoplasmic domains and ten transmembrane segments, and a glycosylated ?-subunit plus a small regulatory protein belonging to the FXYD family. Missense mutations in the genes encoding the a2- and a3-isoforms of the a-subunit, expressed in brain, have been implicated in three neurological disorders, hemiplegic migraine type 2 (HM2), rapid-onset dystonia parkinsonism (RDP), and alternating hemiplegia of childhood (AHC). In 2009 we published together with John Roder’s group in Toronto in Proc. Natl. Acad. Sci. USA a mouse model (“Myshkin”) showing a mutation in a3 Na+,K+-ATPase to give rise to seizures and epileptiform activity in hippocampal brain slices. Thus, there is no doubt that the Na+,K+-ATPase, besides being an indispensable housekeeper in all mammalian cells, in addition is a central player in relation to brain function and pathophysiology.

In 2007 we published in Nature the first paper describing the structure of the Na+,K+-ATPase. An intriguing feature is the location of the C-terminus between the transmembrane segments of the a- and ?-subunits. By mutagenesis experiments we have revealed that the C-terminus regulates the affinity for Na+, a finding published in 2009 in J. Biol. Chem. as Paper of the week. This has set the agenda for studies attempting to elucidate the mechanism by which the C-terminus works. We have also pinpointed the importance of the C-terminus in neurological disease, as two different C-terminal mutations found in RDP patients were shown to reduce the Na+ affinity conspicuously.

Research interests

From our results it may be deduced that a Na+ specific third Na+ binding site, which is distinct from the two sites that alternatingly bind Na+ and K+, is located somewhere in the vicinity of the C-terminus. We are currently investigating the structure-function relationship of this Na+ site.

A number of neurological disease mutants are being characterized in our lab, both at the molecular and cellular levels. Our finding of reduced Na+ affinity in RDP mutants has led to the hypothesis that a rise in intracellular Na+ is an important player in the pathophysiology of the disease. We are testing this hypothesis by studying the effect of Na+,K+-ATPase disease mutations on the intracellular Na+ and K+ concentrations. 

Together with a consortium of European laboratories led by professor Felix Beuschlein, Ludwig-Maximilians-Universität München, Germany, we have recently published an article in NatureGenetics showing that mutations in the Na+,K+-ATPase a1-enzyme expressed in adrenal cortex cause aldosterone producing adenomas (APAs, Conn’s syndrome). Due to the effects of aldosterone excess on Na+ and K+ homeostasis, patients with Conn’s syndrome have hypertension and muscle weakness. In a collection of 308 APAs, 16 (5.2%) somatic loss-of-function Na+,K+-ATPase mutations were identified. The functional disturbance of the Na+,K+-ATPase in the adenoma cells leads to depolarization and Ca2+ influx as well as reduced Na+/Ca2+ exchange, resulting in a rise of the internal Ca2+ concentration and release of secretory vesicles containing aldosterone. Intriguingly, all the Na+,K+-ATPase mutations identified in the APAs are located in the ion binding domain of the protein and affect the functionality of the glutamic acid residue of transmembrane segment M4 in K+ binding. In the figure is shown the Na+,K+-ATPase structure, highlighting the transmembrane region. One of the mutations identified in the APAs replaces Leu104 with Arg (see DNA sequencing results in figure). This leucine holds the glutamate of M4 in correct position for binding of the K+ ion, and the large positive arginine interferes with this function. Our results demonstrate a strongly reduced sensitivity of the mutant Na+,K+-ATPase to K+ activation of dephosphorylation, indicating defective K+ binding. Hence, in the presence of 1 mM K+ the wild type phosphoenzyme level is only 13%, whereas in the mutant a high phosphoenzyme level of 91% is seen, despite the presence of K+ (column diagram).   

Methodologies 

Eukaryotic expression system: Stably and transiently transfected COS cells. A ouabain selection technique is used for isolation of stable COS cells expressing wild type and mutant Na+,K+-ATPase protein at a level as high as 70 pmol functional enzyme per mg total membrane protein. The high expression level allows a detailed functional characterization of the mutants in kinetic studies.

Na+,K+-ATPase purification: purification of Na+,K+-ATPase from pig kidney by differential centrifugation and sucrose gradient centrifugation in a special set-up using a zonal rotor. The high level of purification obtained allowed for the crystallization experiments leading to the Nature paper in 2007.

Protein analysis: Western blotting, Na+,K+-ATPase activity measurements, kinetic studies of conformational changes, rapid-kinetic measurements of the rate of phosphorylation and dephosphorylation at millisecond time scale, ATP synthesis by reversal of the enzyme cycle (“ADP-ATP exchange” activity), determination of Na+  and K+ affinities and occlusion of Na+ and K+. These measurements can be combined to provide information about each partial reaction step in the transport cycle of the Na+,K+-ATPase.

Cell physiology: Rate of cellular uptake of radioactively labeled Rb+ (K+ congener), determination of the intracellular concentrations of Na+ and K+ by use of flame atomic emission spectrometry.

RNA and DNA analysis: site-directed mutagenesis, design of siRNA, cDNA synthesis, PCR analysis, DNA purification

Collaborators and centres (selected)

  • Professor John Roder, Mount Sinai Hospital, Toronto, Canada
  • Professor Felix Beuschlein, Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, Germany
  • Professor Richard Warth, Medizinische Zellbiologie, Universität Regensburg, Germany
  • Professor Paolo Mulatero, Department of Medical Sciences, University of Torino, Italy
  • Professor Chikashi Toyoshima, Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan
  • Professor Gustavo Blanco, Molecular and Integrative Physiology, Kansas University Medical Center, USA
  • Professor Maria-Jesus Sobrido, Universidad de Santiago de Compostela, Spanien
  • Professor Henrik Callesen, Department of Animal Science, Aarhus University, Denmark
  • Senior Scientist Emøke Bendixen, Department of Animal Science, Aarhus University, Denmark
  • Associate professor Knud Larsen, Department of Molecular Biology and Genetics, Aarhus University, Denmark
  • Professor Flemming Cornelius, Department of Biomedicine, Aarhus University, Denmark
  • Professor Helle Prætorius, Department of Biomedicine, Aarhus University, Denmark
  • Associate professor Mads S. Toustrup Jensen, Department of Biomedicine, Aarhus University, Denmark
  • Professor Jens Peter Andersen, Department of Biomedicine, Aarhus University, Denmark
  • Professor Torben Clausen, Department of Biomedicine, Aarhus University, Denmark
  • Interdisciplinary Center for Membrane Proteins: MEMBRANES at Aarhus University, Denmark

Research group members

Group leader

Bente Vilsen

Professor
M
H bygn. 1160, 327
P +4587167736
P +4523822977

Publications

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Revideret 20.09.2017