Ricardo Miledi - US grants

Serotonin (5HT) and its receptors are part of a major neurotransmitter system, whose malfunction is implicated in a variety of human diseases. To understand the malfunction, and possibly alleviate its consequences, we need to know more about the receptors on which 5HT acts. For this purpose a multidisciplinary study will be made of the synthesis, structure and function of 5HT receptors, using frog oocytes and recombinant DNA research. 5HT receptors will 'transplanted' from the brain into the membrane of Xenopus oocytes by isolating messenger RNA, from rat and human brains, and injecting it into the oocytes. Translation of the foreign mRNA by the oocyte causes it to acquire functional 5HT receptors. Electrophysiological techniques will be used to study the ionic membrane channels opened, or closed, by 5HT action and the involvement of cyclic nucleotides, phosphoinostides and calcium ions as channel operators will be investigated with biochemical and electrophysiological methods. Serotonin receptor types will be characterized through pharmacological studies, and a search will be made for selective drugs. This information will help develop compounds that may be used in the treatment of disorders of the 5HT system. Brain mRNA will be the subject of several studies. For example, the receptors and channels expressed by mRNA from the normal and diseased human brain will be investigated for their possible involvement in diseases such as Alzheimer, Cushing and Huntington; and the ontogenetic development of 5HT receptors will be studied by isolating mRNA from the developing brains and expressing it in oocytes. To determine the structure of 5HT receptors two approaches will be used: purification of the receptors and gene cloning. Production of monoclonal anti-receptor antibodies and of synthetic oligonucleotide probes will help greatly in cloning the genes. However, the receptor/channel cloning work will start even before these are produced. mRNA preparations will be fractionated to separate mRNAs coding for different receptors and channels. The partially purified mRNA fractions coding for 5HT receptors and chloride channels will be used to construct cDNA libraries that will be screened by various procedures, including functional assays in oocytes.

The discovery of a new substance, found in the blood serum of many vertebrates, including humans, has been purified and preliminary biochemical evidence indicates that the factor is a special type of albumin. The factor activates a very general intracellular messenger system which is involved in many vital processes. Studies show that this serum factor inhibits the multiplication of cancer cells and also causes the retraction of nerve cell processes. The substance is not present in normal cerebrospinal fluid, but it is present in cerebrospinal fluid of patients following neurotrauma. This factor may play a role in injury and repair of the nervous system. The main goal of this project is to determine the amino acid sequence and structure of the active site of the serum factor using enzymatic and chemical fragmentation of the factor combined with methods of protein purification and structural analysis. Blood serum and albumin are widely used in medicine and in tissue culture. Gaining more knowledge about this factor will have important implications for both basic and medical sciences.

Neuronal inhibition is fundamental to brain function, and in vertebrates, the major inhibitory neurotransmitter is gamma-aminobutyric (GABA). Dysfunction of GABA-ergic systems appears to be involved in the development of numerous neurological and psychiatric diseases such as epilepsy, myoclonus, depression and anxiety, and might also play roles in Huntington's disease, schizophrenia and alcoholism. In mammals there are two well characterized families of GABA receptors. GABA-A receptors are ligand-gated Cl- channels, which are specifically antagonized by the convulsive alkaloid bicuculline. GABA-B receptors couple to GTP-binding proteins and intracellular messenger pathways, and are stereospecifically activated by (-)baclofen. GABA-A receptors appear to be the site which mediates actions of therapeutically useful drugs such as benzodiazepines and barbiturates, together with some of the sedative effects of alcohol. Drugs which selectively interact with GABA-B receptors are presently being developed, and could be of value in treating spasticity, seizure, and depression. Using RNA expression studies in frog oocytes, we have detected a novel class of mammalian GABA receptor, with pharmacological and electrical properties which clearly distinguish it from GABA-A or GABA-B. This receptor has high affinity for GABA, but is insensitive to both bicuculline and baclofen, and is not modulated by benzodiazepines or barbiturates. In view of the importance of other classes of GABA receptor in the CNS, we propose to characterize the structure, pharmacology and electrical properties of these novel GABA receptors. This will be a preliminary step in developing drugs which interact specifically with this site, and will provide a foundation for understanding the physiological roles played by bicuculline/baclofen-insensitive GABA receptors in mental function and health.

Serotonin (5HT) and its receptors are part of major neurotransmitter pathways in both the central and peripheral nervous systems. It is therefore important to know more about the receptors on which 5HT acts and the channels which they operate. for this purpose a multidisciplinary study will be made of the structure and function of 5HT receptors, using frog oocytes and recombinant DNA research. This will provide information about the neurotransmitter receptors and associated channels at all levels, from their genes to their mode of action. 5HT receptors will be 'transplanted' from the brain into the membrane of Xenopus oocytes by isolating messenger RNA from rat and human brains, and injecting it into the oocytes. Translation of the foreign mRNA by the oocyte causes it to acquire functional 5HT receptors, where they are made more amenable to study than if they were in their native brain cells. Furthermore, Xenopus oocytes are extremely sensitive detectors of mRNAs coding for neurotransmitter receptors and channels. Therefore, oocyst will be used for cloning the genes of the receptors and channels involved in the action of 5HT on brain cells. This will be done by a novel approach that avoids the difficult task of having first to purify the proteins. 5HT receptors, transplanted from the brain into the oocyte membrane, mediate at least three different types of responses; possibly involving different receptors. Two of the 5HT responses are caused by the opening of different types of Cl- channels, while the other is caused by the closing of K+-channels. Electrophysiological, biochemical and immunological techniques will be used to study the 5HT receptors and associated channels, as well as the intracellular receptor-channel coupling systems operated by 5HT. Apart from the Cl- channels activated by 5HT, a different Cl- channel is expressed in oocytes by mRNA from Torpedo electrocytes. This mRNA will be used to clone the electrocyte channel, and the clones obtained will then be used to clone other Cl- channels of brain cells. Chloride channels are a very important element in central and peripheral neurons as well as in muscle, secretory and epithelial cells. Accordingly 5HT receptors and Cl-channels have been implicated in a wide variety of human diseases; e.g., mental depression, schizophrenia, Down's syndrome, Cushing's, Huntington and Alzheimer diseases, myotonia and cystic fibrosis. Therefore, this study aims at cloning 5HT receptors and Cl- channels to study their structure and function because the results will undoubtedly help to understand and alleviate some human diseases.

Serotonin (5HT) and its receptors are part of major neurotransmitter pathways in both the central and peripheral nervous systems. It is therefore important to know more about the receptors on which 5HT acts and the channels which they operate. for this purpose a multidisciplinary study will be made of the structure and function of 5HT receptors, using frog oocytes and recombinant DNA research. This will provide information about the neurotransmitter receptors and associated channels at all levels, from their genes to their mode of action. 5HT receptors will be 'transplanted' from the brain into the membrane of Xenopus oocytes by isolating messenger RNA from rat and human brains, and injecting it into the oocytes. Translation of the foreign mRNA by the oocyte causes it to acquire functional 5HT receptors, where they are made more amenable to study than if they were in their native brain cells. Furthermore, Xenopus oocytes are extremely sensitive detectors of mRNAs coding for neurotransmitter receptors and channels. Therefore, oocyst will be used for cloning the genes of the receptors and channels involved in the action of 5HT on brain cells. This will be done by a novel approach that avoids the difficult task of having first to purify the proteins. 5HT receptors, transplanted from the brain into the oocyte membrane, mediate at least three different types of responses; possibly involving different receptors. Two of the 5HT responses are caused by the opening of different types of Cl- channels, while the other is caused by the closing of K+-channels. Electrophysiological, biochemical and immunological techniques will be used to study the 5HT receptors and associated channels, as well as the intracellular receptor-channel coupling systems operated by 5HT. Apart from the Cl- channels activated by 5HT, a different Cl- channel is expressed in oocytes by mRNA from Torpedo electrocytes. This mRNA will be used to clone the electrocyte channel, and the clones obtained will then be used to clone other Cl- channels of brain cells. Chloride channels are a very important element in central and peripheral neurons as well as in muscle, secretory and epithelial cells. Accordingly 5HT receptors and Cl-channels have been implicated in a wide variety of human diseases; e.g., mental depression, schizophrenia, Down's syndrome, Cushing's, Huntington and Alzheimer diseases, myotonia and cystic fibrosis. Therefore, this study aims at cloning 5HT receptors and Cl- channels to study their structure and function because the results will undoubtedly help to understand and alleviate some human diseases.

0082789
Miledi

All the functions of the brain, functions such as our sensations, love, hate etc. depend, in the last instance, on the transfer of signals from one nerve cell to another. The main process of synaptic transmission of signals involves the release of a chemical substance (neurotransmitter) from one neuron and its action on neurotransmitter receptors located in the neighboring nerve cells. Essentially, the interaction of a neurotransmitter with its receptor either stimulates or inhibits the neuron. The main inhibitory neurotransmitter in the vertebrate brain, including that of humans, is a simple chemical called ?-amino butyric acid (GABA). This neurotransmitter was known to act on two types of GABA-receptors (GABAA and GABAB); and a few years ago a new type of GABA receptor was discovered (GABAC).
The main purpose of this research is to determine the structural and functional characteristics of GABAC receptors, to determine their localization in the brain, and to search for new members of the GABAC family of receptors. The basic scientific information obtained will lead to a better understanding of how the brain works and will also serve as a first step towards the rational design of new medicinal drugs, targeted specifically to GABAC receptors.