Most people have most likely experienced unpleasant muscle cramps. These cramps are the result of the action of P-type ATPases. During a muscle cramp, the muscles contract, i.e. they tense and remain in this state, resulting in pain. The reason for this is that the Ca-ATPase in the muscle cells does not work properly due to a lack of magnesium ions. The ATP molecules, which are the “fuel” of Ca-ATPase, are only effective in combination with magnesium ions. This means that only functional Ca-ATPases are able to transport calcium ions back into the cell stores causing the muscles to relax.

ATPases are found in all organisms and are proteins that cleave ATP into ADP (adenosine diphosphate) and phosphate, a reaction which leads to the release of energy that can be used by the enzyme to fuel other reactions. For example, ATPases also deliver the energy that is required for the transduction of signals. Some of the ATPases are membrane proteins which transport ions between different cell compartments or between cells and the extracellular space (ion pumps). The impairment of membrane protein function can lead to severe metabolic disorders or to the loss of organ function. Cells cannot live without APTases, which are active ion transporters and maintain thermodynamic disequilibria. Thermodynamic equilibrium corresponds to the death of the cell.

The P-type ATPases are a subgroup of ATPases and are at the very centre of Professor Apell’s research. This class of ATPases is temporarily phosphorylated when it reacts with ATP. The P-type ATPase family includes the Ca-ATPase in the muscle cells, which leads to the relaxation of muscle fibres, the Na-K-ATPase and the H-K-ATPase. Na-K-ATPases are present in all animal cells and are absolutely vital for cell survival. The intoxication of Na-K-ATPase leads to the very rapid death of a cell. In nature, there are toxins that can block Na-K-ATPase, resulting in the paralysis or death of prey, or which serve to protect an organism against predators. The digitalis toxin, for example, blocks Na-K-ATPase. Gastric H-K-ATPase occurs in specific cells of the stomach mucosa and controls the secretion of hydrochloric acid by pumping H+-ions (protons) into the stomach. In people suffering from stomach mucosa diseases or gastric ulcers, the production of acid can be reduced by inhibiting the H-K-ATPase enzyme.

Hans-Jürgen Apell’s team is focused on the search for information about the molecular mechanism of the active transport of ions through the cell membrane by way of P-type ATPases. These insights might, for example, also contribute to the development of new and more effective inhibitors for the treatment of stomach acid secretion. “The methods used enable, amongst other things, the introduction of assays with which it is possible to test any pharmaceutical for its effect on P-type ATPases,” said Hans-Jürgen Apell. In order to achieve their goal, the researcher and his team are mainly interested in elucidating the ion transport of P-type ATPases. Some fundamental principles are already known.

“The transport of ions through the enzyme Na-K-ATPase happens in a ping-pong mode: At first, the sodium ions of the cell’s interior are bound by the ion binding sites, they are then transported through the channel and released on the other side of the cell membrane. The potassium ions then bind to the same binding sites and are transported into the cell,” said Hans-Jürgen Apell describing the transport of sodium and potassium ions across the cell membrane. Similar mechanisms have also been found for Ca-ATPase and H-K-ATPase. Structural and kinetic experiments are necessary to investigate the cyclic course and ping-pong mode in greater detail. “The precision and speed with which these ion pumps transport the ions is fascinating. The pumps consist of only 1,000 amino acids. We would like to understand how this speedy transport is possible as well as finding out about the molecular mechanisms underlying the ion transport,” said Hans-Jürgen Apell.

Professor Apell’s major research focus concentrates on the investigation of membranes obtained from animal cells. “We use membranes from rabbit kidneys (for the enzyme Na-K-ATPase), rabbit muscles (Ca-ATPase) and the pig stomach (H-K-ATPase),” said Apell. The animal ATPases and human ATPases only have a few differences in their amino acid composition, so that no significant differences between the animal and human proteins are expected and have so far not been detected.

Apell’s experimental investigations on the mechanism of ion pumps involve mainly optical methods. Electrochromous fluorescent dyes enable the observation of ion movement in the ion pumps with extremely high temporal resolution (in the microsecond range). The researchers use functionally active, pure preparations of the three P-type ATPases. Using commercially available or self-made fluorescence spectrometers and measurement devices, the researchers are investigating the behaviour of the ion pumps when the ion or ATP concentration is altered. “The measured changes in fluorescence are then analysed and this enables conclusions to be drawn on the ion movements in the ion pumps,” said Hans-Jürgen Apell.

This method has already led to a range of findings that could not be obtained with electrophysiological investigation methods. The fluorescence method used enables the measurement of charge movements, which are very slow or only cover short membrane stretches or which cannot be resolved due to a small signal-noise ratio. When a new binding state is reached, the fluorescence signal is stable, making it easily and accurately measurable. Electrical signals are of a transient nature. The new method enabled the researchers to clarify what happens when sodium ions are bound on the cytoplasmic side of the Na-K-ATPase, something that was not previously possible using electrophysiological methods. The researchers were also able to find out for the enzyme H-K-ATPase which partial reactions are associated with charge movements and which are not.

The researchers also use mathematical modelling of the experimental data, which applies different mechanistic approaches to create well-defined conditions for theoretical model descriptions of P-type ATPases. The researchers are also hoping to discover a general ion pumping mechanism of P-type ATPases. In these investigations, Apell’s research team is working closely with research groups in Australia, Israel, Russia and the USA, in which the cooperating teams use the methods established in the respective laboratories. This synergistic work saves considerable amounts of money as the experimental set-ups are usually very complex.

The 3D structures of proteins in atomic resolution prepared with computers play an important role in obtaining detailed insights into ion transport. “This helps to obtain a spatial idea of the different (functional) parts or domains of proteins, for example to gain some idea of the location of the ion binding sites, their access or the sites where a toxin can bind and block or modify a protein.”

The highly pure proteins prepared by Apell’s team are also of great interest for diverse investigations in pharmaceutical research. “For example, the measurement methods can also be used to clarify the interaction between pharmacologically active substances and P-type ATPases in order to identify the effect of the substances and clarify the interaction mechanism. The researchers have previously worked in this field with Altana Pharma (now Nycomed) in Constance in order to investigate the interaction of Altana’s Pantoprazole H-K-ATPase blocker with Na-K-ATPase,” said Hans-Jürgen Apell.

One of Apell’s major future research goals is to investigate the differences between the different isoforms of an Na-K-ATPase which his colleagues from Israel succeeded in expressing in yeast cells. The researchers now hope to clarify the molecular causes of this. “This fundamental knowledge is certain to be very interesting for pharmacological research, because efforts have been concentrated for a long time on finding drugs targeting Na-K-ATPase for the treatment of cardiac insufficiency.”

Another major aim of Professor Apell’s research group is the investigation of the molecular mechanisms through which chemical energy from the hydrolysis of ATP is converted into “transport energy”. “We still do not have a concrete idea as to how this works, but it is extraordinarily interesting because the degree of efficiency is between 60 and 90 per cent, something that cannot be achieved with macroscopic machines.”

mst – 6 November 2008
© BIOPRO Baden-Württemberg GmbH

More Information:
Prof. Dr. Hans-Jürgen Apell
Universität Konstanz
Fachbereich Biologie
Arbeitsgruppe Membranbiophysik
Tel.: +49 (0)7531 88-2253
E-Mail: h-j.apell@uni-konstanz.de