Ca-ATPase & H,K-ATPase: Structure, transport mechanism, and pharmacological aspects

Group leader: prof. emeritus Jesper Vuust Møller

Three-dimensional structure showing Ca2+-ATPase as consisting of 10 helical membrane embedded segments (M1-M10) with one of the two bound calcium ions (shown as a red ball) and three cytosolic domains (A, P, and N). The arrows on the simplified sketch of Ca2+-ATPase on the right side shows the location of proteolytic splits. Regions reacting with sequence specific antibodies outside the membrane are shown in green.

http://www.biophys.au.dk/Person/jvmbj/images/dias1.JPG

Initiation of muscle contraction requires activation of the myofibrils by Ca2+ released from the Ca2+ stores in the sarcoplasmic reticulum. The function of the sarcoplasmic reticulum Ca2+-ATPase is to transport Ca2+ back from the cytoplasm to the inside of the sarcoplasmic reticulum during the relaxation phase of the muscle contraction cycle. The protein performs this job efficiently at the expense of ATP hydrolysis, which leads to Ca2+ concentrations 103-104 times higher on the inside than on the outside of the membrane. Much is known about the structure of the protein so that it is now possible to use this ATPase to explore in a fundamental way mechanisms involved in converting an enzymatic reaction (ATP hydrolysis) into a vectorial transport process of calcium ions across the sarcoplasmic reticulum as explained below.

In the three-dimensional structure of the protein shown above, which has been obtained by X-ray diffraction of Ca2+-ATPase crystals we see that the ATPase consists of one membraneous and three cytoplasmic domains: a nucleotide (N), a phosphorylation (P), and an A (actuator) domain. The membraneous domain consists of ten (M1-M10) membrane spanning helical segments that harbour in their interior two well-defined Ca2+ binding sites. Our studies at the moment focus on (i) how the uptake of two calcium ions from the cytoplasm to the intramembraneous binding sites takes place, (ii) the active transport of the two Ca2+ from the intramembraneous binding sites across the membrane, and (iii) the coupling between these processes and ATP hydrolysis (formation of an energy rich phosphorylated E~P intermediate, followed by Ca2+transport and dephosphorylation).

We study these questions by a variety of biophysical, biochemical, and immunological techniques. This entails the use of proteolytic enzymes for dissection and identification of resulting proteolytic fragments with antibodies against selected Ca2+-ATPase regions (sequence specific antibodies). We also frequently use detergents either as a substitute of membrane lipid or for preparation of transport competent lipid vesicles with incorporated ATPase. As an example we have found with these techniques that by cleaving the link between the A-domain and the membrane domain (see the red arrow on the sketch above) we can block the transport process without affecting the overall ATPase structure and the first steps of transport. This suggests a central role of the link and the A-domain in the transport of Ca2+ across the membrane (Ref. 6). Other findings (Ref. 5) indicate that the C-terminal part of the ATPase is essential for the first steps of the transport process (binding of Ca2+ and phosphorylation by ATP). By piecing together information of this kind obtained by us, and by others with the aid of other methods, we shall expect to be able to form a coherent picture of the processes leading to active transport of Ca2+.

Persons (pre- and postgraduate students) interested in performing investigations along these lines at an institute focusing on studies on P-type ATPases are welcome to contact us.