Much of our work revolves around a novel technique that allows the functional expression of receptor and channel proteins from the messenger RNAs that encode their structures. Detailed knowledge of receptors and channels in the brain is at present meager. To a large extent, this is due to the small size and inaccessibility of neurons in the brain, particularly in the human brain. It seemed to us that these problems could be avoided if it were possible to induce larger cells to acquire the receptor and channel proteins that occur in brain neurons. For this purpose, we chose the oocytes of the frog Xenopus laevis, which are over 1 mm in diameter and can easily be micro-injected with foreign messenger RNA. Following injection of brain messenger RNAs (including those from humans), the oocyte expressed receptors and channels that could be activated by application of drugs or by changes in membrane potential. In this way, we have “transplanted” many of the known brain neurotransmitter receptors to the oocyte, including receptors to acetylcholine, catecholamines, excitatory and inhibitory amino acids, serotonin, and peptides. Once expressed in the large oocyte cells, the study of receptor function becomes much easier. For example, we use such techniques as voltage clamp, noise analysis, and patch clamp recording to examine the currents flowing through the ion channels activated by receptor binding, and to see how these currents are modified by clinically useful drugs. Another topic that becomes more amenable for study is the role of intracellular messengers, which are used in several neurotransmitter systems as the link between receptor binding and activation of membrane channels. In the oocyte, it is possible to monitor putative second messengers s uch as calcium and cyclic nucleotides during activation, and also to examine the effect of injection of these substances into the cell. A different application is to use the oocyte as a screening system to test and develop new drugs, and to measure the extent to which messenger RNAs coding for different neurotransmitter receptors are expressed in different tissues. For example, messenger RNAs are being isolated from different areas of the brain, from brains at different stages or embryological development, or following various pathological conditions such as Alzheimer’s disease.
Lab MembershipsMiledi Lab (Principal Investigator/Director)
Laboratory of Cellular and Molecular Neurobiology
Department of Neurobiology and Behavior
University of California
Web Page: http://faculty.sites.uci.edu/rmiledi/