All sluices have gates that control the amount of water being channelled through. Similar gates are also found in the ion channels of neurons and other cell types. The controlled transport of calcium across cell membranes is crucial for many biological processes, including the transfer of information into the brain, kidney function and the rhythmic activity of the heart muscle. A team led by Prof. Dr. Norbert Klugbauer at the University of Freiburg is focused on elucidating the function of calcium channels, the proteins that regulate them and the effects on learning and memory. The researchers are investigating the molecular mechanisms and electrical properties of calcium channels as well as using mice to gain insights into calcium-dependent memory. At the moment, they are concentrating on a channel type that was discovered in plants.
Calcium is one of the most important ions in the human body. It is essential for maintaining bone mass as well as for the transmission of information between nerve cells or in muscles. Information is transferred by way of ions transported across the cell membrane or across the membranes inside cells, such as those in the endoplasmic reticulum. The channels consist of different protein subunits. Activation of the ion-conducting pores allows calcium ions to enter the cell. The channels are multi-subunit complexes that can remain open for different lengths of time; the opening and closing of the channels also depends on the interaction between the channel proteins and other regulatory proteins.“How does this regulation work, which interaction partners are involved?” asks Prof. Dr. Norbert Klugbauer from the Institute of Experimental and Clinical Pharmacology and Toxicology at the University of Freiburg. “And what consequences does the regulation of the channels have on the physiological and behavioural level?”
All these questions imply that Klugbauer’s team is using a broad range of different methods in their search for answers. Ion channels are an excellent example of molecules that play an important role on almost all biological levels. This is because molecular changes have an immediate effect on the electrophysiological properties of nerve cells, which in turn also affects processes such as learning and memory. An example of the experimental procedures that are carried out by Klugbauer’s team is their work on P/Q type calcium channels, voltage-dependent calcium-conducting pores found in the nerve cells of the hippocampus, a brain region associated with learning and memory. Klugbauer and his colleagues were the first researchers in the world to switch off the genes coding for these channels in specific tissues, i.e. in the hippocampus and the neocortex. How do mice that lack P/Q calcium channels in these regions act? Can they remember the position of an escape platform in a water tank, for example?
Normal mice tend to swim around aimlessly when they are released into a water tank with an escape platform for the first time. However, on subsequent trials where the platform remains in the same position, the mice are able to locate the platform more quickly each time. However, knock-out mice that lack specific calcium channels swim around aimlessly on the first and all subsequent trials. “This clearly shows that P/Q type calcium channels located in the membranes of specific hippocampus cells play a key role in spatial memory,” said Klugbauer. In order to further investigate the role the channels play, Klugbauer and his team are now working to identify the proteins with which the membrane channels interact. They are working with the neurophysiologist Prof. Dr. Bernd Fakler and the biochemist Dr. Uwe Schulte from the University of Freiburg, both specialists in the field of proteomics who also use the so-called split-ubiquitin technique, one of the many technical skills that today’s life scientists have.
The split-ubiquitin method is based on the ability of the N- and C-terminal halves of ubiquitin to form a native-like ubiquitin. Ubiquitin has numerous functions, the most important being its attachment to proteins that are defective or unneeded and labelling them for destruction. The split-ubiquitin method can be used to attach ubiquitin to the particular calcium channel under investigation: the researchers couple one half of the ubiquitin molecule to an ion channel and the other half to a selected protein to find out whether it interacts with the channel. When the channel and the protein interact, they move towards each other in the membrane. As a result, the two ubiquitin halves come within close spatial vicinity and merge into a functional protein. This in turn tells the cleavage enzyme to cut off the signalling molecule that was previously attached to the channel protein, the signalling molecule enters the cell nucleus where it activates a reporter gene. Commonly used reporter genes involve fluorescent and luminescent proteins. One such gene makes cells appear blue, which in turn indicates whether a channel has interacted or not with a particular protein under investigation.
“In addition to these experiments, we can also use electrodes to record the electrophysiological characteristics of a particular channel,” said Klugbauer. “How do the strength or the flow of current change when the channels interact with the protein? Do they open more slowly or more quickly? Do they remain open for a longer or shorter period of time?” Klugbauer and his colleagues recently discovered a very promising calcium channel interaction partner and are now taking a closer look at it. In addition, the researchers have over the last year been concentrating specifically on a new type of channel, which until recently appeared to be only present in plants. This calcium channel, a so-called TPC1 channel (TPC stands for two-pore channel), is only half the size of its relatives and has recently been discovered in mammalian cells in the membrane of small vesicles where it controls the influx of calcium into the cells. NAADP (nicotinic acid adenine dinucleotide phosphate) is a calcium ion-mobilising second messenger which medicates its effect by activating TPCs. TPCs located in blood vessel cells are also assumed to play a role in the regulation of blood pressure.
The largest number of TPC1 channels has been found in kidney cells. “We assume that the channel’s role is associated in some way with the function of nephrons, which would mean that it is involved in the concentration and composition of urine,” said Klugbauer. Klugbauer and his team are now using their methods to investigate the renal TPC1 channel and have plans to work with research groups led by Dr. Köttgen and Dr. Kühn from the Department of Nephrology at the Freiburg University Medical Centre in order to clarify the following questions: how do the electrolytes of the urine and other physiological parameters in the kidney change when the channels in the renal tissue are switched off? Do TPC1-deficient mice drink more water than healthy mice? With which proteins does the channel interact? The work related to solving these questions is very much in its early days. The experiments being carried out by Klugbauer’s team are not just of importance for basic research. It has recently been shown that the deposits in the brains of Alzheimer’s patients can also bind to P/Q type calcium channels. Do the memory disturbances of Alzheimer’s patients have something do with these interactions? “Our ultimate goal has always been to find an answer to the question as to whether the channels we are investigating might be therapeutic targets that can be used for the treatment of diseases,” said Klugbauer.
Further information:Prof. Dr. Norbert Klugbauer Experimental and Clinical Pharmacology and ToxicologyDept. IUniversity of FreiburgAlbertstraße 2579104 Freiburg i. Brsg.Tel.: +49 (0)761/ 203 5310E-mail: n.klugbauer(at)pharmakol.uni-freiburg.de