Jens Peter Andersen Lab


We study the mechanism and regulation of membrane pumps and the pathophysiology of diseases caused by pump mutations. JPA has published more than 100 articles about the sarcoplasmic reticulum Ca2+-ATPase (SERCA). This P-type ATPase carries out active transport of Ca2+ from the cytoplasm to the luminal side of sarco(endo)plasmic reticulum to build up a Ca2+ concentration gradient of at least 1000 fold. Two Ca2+ ions are transported for each ATP being hydrolyzed. During the transport cycle the enzyme becomes phosphorylated from ATP at a conserved aspartate in the cytoplasmic domain and undergoes a conformational change from the Ca2+ binding E1 form to the E2 form expelling bound Ca2+ to the lumen, before it dephosphorylates. Important challenges are to find out how and where in the protein the Ca2+ ions are transported through the membrane, and how the movement of Ca2+ is coupled to ATP hydrolysis. In addition the Ca2+ pump has a number of regulatory features and relevance for cellular Ca2+ signaling and pathophysiology. The major method used in the Andersen lab is functional analysis of mutant Ca2+ pumps produced by site-directed mutagenesis. This project was started more than 20 years ago as a visiting scientist in David MacLennan’s lab at the Banting and Best Department of Medical Research, University of Toronto, Canada. We have characterized the functional consequences of several hundred different mutations of the Ca2+-ATPase (see figure). To this end we have developed enzyme kinetic methods that allow determination of the rates of each of the partial reaction steps of the Ca2+ transport cycle as well as the affinity for Ca2+, ATP, and other ligands such as the inhibitors thapsigargin and vanadate, using only the minute amounts of wild-type and mutant Ca2+-ATPase protein that can be harvested from transfected mammalian cells. By functional analysis of mutants we pinpointed the amino acid residues making up the two Ca2+ sites in the protein by mutagenesis, and we demonstrated the separate roles of the individual residues in binding each of the two Ca2+ ions in a sequential mechanism. We identified the ATP binding site and the thapsigargin binding domain, as well as a number of residues of crucial importance for the E1-E2 conformational transition of the phosphorylated protein that moves Ca2+ and simultaneously changes the catalytic specificity at the ATP site from kinase to phosphatase specificity. By mutagenesis we also demonstrated the involvement of the glutamate of the TGES motif in catalysis of the dephosphorylation of the enzyme. In 2009 in a paper selected as Paper of the Week in the Journal of Biological Chemistry, we showed the length of the linker segment connecting the cytoplasmic A-domain with the third transmembrane segment to be a crucial determinant of the rate of the E1-E2 conformational change, thus indicating that strain in the linker built up during phosphoryl transfer from ATP to the protein is a driving force in the rotation of the A-domain leading to energy transduction (“spring model”). We have used the same kinetic methods to characterize the functional differences between the SERCA1, 2, and 3 isoforms and differences beween the SERCAs and the secretory pathway Ca2+-ATPases (SPCA) expressed in cell culture.

In 2010 we started in collaboration with the group of R.S. Molday at University of British Columbia, Vancouver, Canada, to apply our methodology to elucidate the transport mechanism of another P-type ATPase, the phospholipid flippase ATP8A2 (see further below under Research Interests). Our first flippase paper, demonstrating the ability of ATP8A2 to form a phosphoenzyme sensitive to phosphatidylserine and identifying a lysine residue crucial to the interaction with phosphatidylserine, has recently been published in Proc. Natl. Acad. Sci. USA.

Research interests

Ongoing projects on the Ca2+-ATPase include a mutational analysis of the mechanism by which the additional transmembrane segment of the SERCA2b isoform causes the higher Ca2+ affinity of this isoform relative to the other SERCA isoforms. This is a collaborative project with the group of Peter Vangheluwe, K.U. Leuven, Belgium. We are also deeply enganged in defining the mechanism by which ATP in addition to undergoing hydrolysis as a substrate in the phosphorylation reaction modulates the rates of some of the conformational transitions in the Ca2+ transport cycle without being hydrolyzed, and we continue to elucidate the pathway for Ca2+ migration through the enzyme, now with special focus on locating the luminally oriented Ca2+ leaving site.

Phospholipid flippase
As mentioned above, we have recently become interested in a newly discovered group of membrane pumps called P4-ATPases or “flippases” that transport (“flip”) aminophospholipids from the outer part of the cell membrane lipid bilayer to the inner part, thereby creating the asymmetric lipid distribution that enables vesicle formation (endocytosis and exocytosis), fertilization, cell division, as well as apoptosis. The amino acid sequence homology suggests that the overall structure and domain topology of P4-ATPases is similar to that of the catalytic subunits of the Ca2+-ATPase and Na+,K+-ATPase. However, little information is available on the actual mechanism of phospholipid transport and how it is coupled with ATP utilization. Do P4-ATPases form a phosphoenzyme existing in two major conformations E1 and E2? Is lipid flipping toward the cytoplasmic leaflet associated with dephosphorylation, like the transport of K+ from the extracellular to the cytoplasmic side by the Na+,K+-ATPase? Is the formation of the phosphoenzyme of flippases activated by a specific substrate being transported, as established for the phosphoenzyme intermediates of Ca2+-ATPase and Na+,K+-ATPase, which depend on the binding of Ca2+ and Na+, respectively? The P4-ATPases constitute as much as 40% of the P-type ATPases in the human genome. Several genetic diseases (neurological like Angelman syndrome and others such as Byler intrahepatic cholestasis) are caused by mutation of a flippase. ATP8A2 is a flippase expressed in brain and testis, and mutations in ATP8A2 have recently been shown to cause severe mental retardation and other neurological problems in humans. Our hypothesis is that this flippase, and likely also other P4-ATPases, functions by means of a mechanism that translocates the aminophospholipid head group in a way similar to the translocation of cations by Ca2+-ATPase and Na+,K+-ATPase. Because the removal of the phospholipid head group from the membrane-water interphase may be energetically more demanding than the movement of the hydrocarbon chains through the membrane, the interaction of the head group with a protein site is likely a crucial part of the flipping mechanism. We are investigating this hypothesis by identifying the functional changes that result from inserting a series of strategically positioned mutations. To this end we express ATP8A2 mutants in mammalian cell culture, purify and reconstitute them in vesicles of defined lipid composition, and assay their function using, among others, variants of the enzyme kinetic methods previously developed. We will work toward a definition of a binding site for aminophospholipid being involved in the lipid flipping, and we will also examine whether the P4-ATPases transport a counter ion in a way similar to the consecutive Na+ and K+ transport by the Na+,K+-ATPase.


Mutations are introduced in cloned cDNA, and the construct is expressed transiently in transfected mammalian cells (usually COS-1 or HEK293T). Membranes containing the mutant are harvested by differential centrifugation, or the protein is purified by immunoaffinity chromatography following detergent extraction. Functional studies are performed on the microsomal preparation or the purified protein reconstituted in lipid vesicles of defined lipid content by dialysis of the detergent solubilized purified protein after addition of lipid. Measurements of the rate of ATP hydrolysis are carried out by colorimetric determination of Pi release. 45Ca2+ uptake in vesicles by Ca2+-ATPase is determined by Millipore filtration. The partial reaction steps in the enzyme cycle are followed by taking advantage of the acid stability of the phosphorylated intermediate formed by reaction with [gamma-32P]ATP or 32Pi. Acid is used to quench (i.e. “freeze”) the phosphorylated state for quantification of the protein-bound radioactive phosphate. Following acid quenching, the 32P-labeled protein is washed by centrifugation and isolated by SDS polyacrylamide gel electrophoresis under acidic buffer conditions. Quantification of the 32P-radioactivity associated with the protein band is performed by quantitative autoradiography (“phosphor imaging”) of the dried gels. Two basically different types of phosphorylation experiments are employed: (i) ligand dependency of steady state or equilibrium phosphorylation, and (ii) transient state kinetics – the enzyme is incubated for sequential time intervals with [gamma-32P]ATP or 32Pi to allow measurement of the rate of phosphorylation, or the dephosphorylation rate is determined by chasing the phosphoenzyme in conditions preventing further phosphorylation. It is possible to design conditions that lead to selective accumulation of each of the four main intermediates in the transport process, E1, E1P, E2P, and E2, at steady state or equilibrium. The transformation of one state to the next in the reaction cycle can then be followed upon addition or removal of a ligand. These experiments are carried out either by hand mixing at 0°C or with the use of a Bio-Logic QFM-5 quenched-flow module for rapid kinetics (Bio-Logic Science Instruments, Claix, France) that allows measurements of rates under more physiological conditions (25°C, ms time scale).

Collaborators and centres

  • Professor R.S. Molday and PhD student Jonathan Coleman, University of British Columbia, Vancouver, Canada
  • Professors Peter Vangheluwe and Frank Wuytack, K.U. Leuven, Leuven, Belgium
  • Professor David B. McIntosh, University of Cape Town, South Africa
  • Professor Bente Vilsen, Department of Biomedicine, Aarhus University
  • Associate professor Mads S. Toustrup-Jensen, Department of Biomedicine, Aarhus University
  • Professor Poul Henning Jensen, Department of Biomedicine, Aarhus University
  • Interdisciplinary Center for Membrane Proteins: MEMBRANES at Aarhus University, Denmark

Research Group Members

Group leader

Jens Peter Andersen

H bldg. 1160, 315
P +4587167725
P +4520434362


Sort by: Date | Author | Title