On-going research activity
In order to uncover the roles of the transporters, we are using state of the art imaging techniques (quantitative electron microscopy and confocal microscopy) to map the precise localizations of individual transporter protein types as well as their splice variants. We combine imaging with stereological measurements and immunochemical measurements of the tissue content of the individual transporter proteins. This enables us to calculate the exact transporter densities at the various locations (number of transporter molecules per square micrometer cell membrane).
Antibodies - The studies require access to highly specific antibodies. We therefore produce a large number of different antibodies which we subject to extensive purification and rigorous testing. (For detailed discussions see: Danbolt et al., 1998, 2001; Holmseth et al., 2005)
Automation - "What a robot can do, a robot should do." We use robotic equipment to minimize manual work (fully automated ELISA assays, development of Western blots and immunolabeling of tissue sections). Advanced database technology is essential for handling the data produced.
Transporter function and structure - Information on transporter localizations and numbers are important, but not sufficient. We also need to know more about the properties of the molecules themselves. How they operate, how they are regulated, and so on. This we do by reconstituting purified transporter proteins into artificial cell membranes with defined composition. We are currently modifying the assays to allow robotic high-throughput screening for drug interactions and influence of lipids and fatty acids.
Transgenic animals - In addition, we produce animals with specific genetic modifications: e.g. the selective deletion of one gene at a certain age in only one population of cells, leaving the rest of the animal normal, and also allowing normal development of the animal until the time of study. This is required in order to be able to distinguish between the primary role of a transporter from those of secondary changes. For instance, if a protein plays important roles both in the brain and for insulin secretion, then deleting it in both places may make it difficult to sort out its roles in both places because disturbed control of insulin may affect the brain and vice versa.
Computer simulations - Because of the fine structure of the nervous system (e.g. the sharpest electrodes used for electrophysiological recording being more than 10 times thicker than synaptic clefts), and because of the dynamic regulation of virtually all tissue components and proteins, the nervous system cannot be measured without changing it. To overcome this problem, we are using the above obtained data to perform computer simulations. Some studies have been performed and more are under way.
Neurological disease - Apart from being interesting in itself, data generated from the above studies are being combined with data from our collaborators specifically to improve our understanding of memory formation, epilepsy, amyotrophic lateral sclerosis (ALS), bacterial meningitis, stroke and Huntington's and Alzheimer's diseases.
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