Flemming Cornelius

Research

Our research is concerned with the structure, function and regulation of P-type ATPases, mainly Na,K-ATPase (NKA) and H,K-ATPase(HKA). We purify preparations from pig kidney (NKA), pig heart (NKA), pig stomach (HKA), and shark salt glands (NKA). The latter has been used as a model tissue for Cl- secreting epithelia in all vertebrates from sharks to mammals.

Research interests

The steady state and pre-steady state kinetics and electrogenic properties of the overall and partial reactions of Na,K-ATPase are investigated in purified and reconstituted P-type ATPases.

Regulation of P-type ATPases by small single-span proteins. Since our discovery and characterization of a new regulatory protein (PLMS, phospholemman-like protein from shark) specifically associated with Na,K-ATPase from the salt gland of the shark it has become clear that the activity of Na,K-ATPase in various species and tissues is finely regulated by interaction with a group of small, single-spanning proteins called the FXYD proteins. Some FXYD proteins exert their effects on the Na,K-ATPase depending on their state of phosphorylation as regulated by protein kinases/phosphatases. We have characterized this protein kinase dependent interaction of FXYD10 with the Na,K-ATPase.   

Regulation of Na,K-ATPase by oxidative signalling. Both FXYD and b are modified by  glutathionylation at specific cysteines. Thus glutathionylation of FXYD reverse glutathionylation-induced inhibition of Na,K-ATPase. The mechanism of this oxidative signaling pathway is investigated.

Investigation of the interactions of phospholipids and cholesterol with the Na,K-ATPase. For this we use reconstitution of Na,K-ATPase into small lipid vesicles (liposomes) of defined lipid composition. Indeed, cholesterol seems to play a crucial role in supporting Na,K-ATPase activity, a fact that has led to the suggestion of specific interaction (binding) of cholesterol with Na,K-ATPase.

Recently we have been engaged in research concerning the molecular 3D structure of Na,K-ATPase in various intermediate states and after binding of cardiotonic steroids. Thus structures of shark Na,K-ATPase in the E2PK2-state and in a state with bound ouabain has been determined at atomic resolution. Interestingly a cholesterol molecule is resolved at an important location in the structure that may explain its significance in Na,K-ATPase function.

Methodologies

  • Protein analysis: Western blotting, immunostaining, activity measurements, functional studies, phosphorylation and dephosphorylation reactions, autoradiography, imaging
  • Biochemical and biophysical methods: stopped-flow fluorescence, quenched-flow measurements, measurement of electrogenic membrane potentials and pH gradients using fluorescent probes, measurements of intermolecular interactions  using molecular cross-linking
  • Structural analysis:  X-ray crystallography

 

 

Collaborators and centres

  • Bente Vilsen
  • Yasser Mahmmoud
  • Anne Lillevang, Dept. Biomedicine, University of Aarhus, Denmark
  • Ole Mouritsen, MEMPHYS, University of Southern Denmark, Denmark
  • Chikashi Toyoshima,  Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan.
  • Helge Rasmussen, Department of Cardiology, Royal North Shore Hospital, University of Sydney, Australia
  • Ron Clarke, School of Chemistry, University of Sydney, Australia

Research group members

  • Anne Mette Bech Sørensen
  • Bianca Franchi

Group leader

Flemming Cornelius

Lektor
M
H bygn. 1182, 109
P +4587167746
P +4529454673

Publications

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