rates of synthesis are altered during the period of serotonin application (the analog equivalent to the training period). Some of these proteins are transiently expressed early in training (early proteins). Others are expressed later (late proteins), that is, the induction of these proteins is blocked by inhibitors of RNA synthesis. Barzilai, Sweatt, and Kandel are now purifying these proteins using preparative two-dimensional gels to begin sequencing these proteins.

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in press.

Channels and Receptors

Brain GABAA and Nicotinic Receptors: Molecular Biology and Molecular Electrophysiology

E.A. Barnard

MRC Molecular Neurobiology Unit, Medical Research Council Centre, Cambridge CB2 2QH, England

Molecular Biology of Brain Receptors: An Agenda

The human nervous system, and in particular the human brain, is far more complex and difficult to understand than any other system that one can select as a target for the chemotherapy of disease. While many drugs active in this system are currently of great pharmaceutical significance―e.g., tranquillisers, neuroleptics, sedatives, analgesics, anaesthetics - they have essentially evolved by trial and error. This is probably true to a greater extent for effects on the central nervous system than for any other sector, considering the special difficulties of access and the incredible complexity of the cellular and hierarchical structure of the brain. The need for therapeutic intervention in this complexity is correspondingly enormous, as is obvious in the immense problems of mental illnesses, dementias, epilepsy, stroke, narcotics abuse, and many others.

Rational drug discovery must start with targets at the strictly molecular level; effects at higher levels of complexity should be considered at subsequent stages of testing. Recent advances in basic neurobiology have disclosed a number of protein types essential for the specialised functions of the nervous system. Of particular importance among these proteins are the receptors for neurotransmitters and hormones, which perform most cell-to-cell signalling in the nervous system.

An important task of neurobiology is now to define the functions of

receptor proteins; to locate them at the cellular, neural pathway, and developmental levels; to clone their genes; to decipher their structures; and to map their interactions. Molecular neurobiology is an emerging multidisciplinary activity that is applying the new technologies of molecular genetics and molecular cell biology to elucidate the detailed architecture of neurones, synapses, and neural pathways. All the information now being assembled is capable of locating potential targets for beneficial drug interventions.

A Macromolecular Data Base for Future Drug Design for the Nervous System

The study of neurotransmitter receptors offers an example of what might be usefully sought. The biological tools to be used include the cloning of their cDNAs, the expression in the Xenopus oocyte of their mRNAs, their transfer to nonneuronal host cells in culture by gene transfection, analysis of singlemolecule receptor events by patch-clamping, plus site-directed mutagenesis and antipeptide monoclonal antibody location to map their topology and their functional determinants. The analytical tools to be applied include the computer-aided interpretation of amino acid sequences, molecular graphics, and computational approaches to the modelling of interactive surfaces and the docking thereat of drug molecules.

Approaches to the three-dimensional structure of receptor drug-binding domains, hitherto completely elusive, are in prospect, from these methods and from advances in biophysical analyses. Important insights into these domains emerge from the recognition of receptor super-classes and homologies and the frequent recurrence of particular mechanisms and structural motifs. These important pieces of information have already been revealed by the cloning of receptor DNAs.

For drug design, the actions of many types of drugs in the nervous system will only become comprehensible when analysed in terms of receptor structure and binding-site docking requirements.

The Brain GABAA And Nicotinic Acetylcholine
Receptors

Our and collaborating laboratories have been working on the cloned cDNAs encoding the GABAA receptor. In this receptor, GABA gates an intrinsic anion channel. The receptor purified from bovine brain contains a and ẞ subunits. cDNAs encoding the a and ẞ subunits of this receptor have been cloned. Pure RNAs encoding the a and ẞ subunits can be generated in vitro from the a and the ß cDNAs; if injected together into the Xenopus oocyte,

these produce the functional receptor and ion channel. We have deduced a model for this receptor.

Multiple subtypes of this receptor are known pharmacologically and have now been identified as arising from different genes. Their structural and functional differences are of interest, as well as their genomic structures and chromosomal locations.

Some of the cDNAs encoding the subunits of neuronal nicotinic acetylcholine receptors have been analysed similarly, again with oocyte expression. These belong to the same super-family. Structural motifs common to both receptor types, and determinants of special functional attributes in each, provide insights into the general principles governing the ion channel-containing receptors.

Potential Applications to Diagnosis and Treatment

The GABAA receptor is the site of benzodiazepine (BZ) action in the brain. Therefore, conditions ameliorated by BZs or by GABA-release activators are candidates for the involvement of this receptor. This brings into focus, among others, some of the epilepsies, manic depression, panic disorders, and possibly autism.

The neuronal nicotinic receptors in the nucleus basalis and in the cortex have been shown to be in deficit in Alzheimer's disease, and cholinergic reinforcement is a preferred goal for present therapy designs there.

In both these cases, the availability of cloned subunit DNAs for the receptors opens exciting new approaches. These include:

1. The human chromosomal localisation of the receptors' genes, and the search for linkage to an inherited form of each candidate disorder.

2. The screening of human pedigrees of candidate disorders, with these DNAs as probes, to seek segregation of polymorphisms at that locus with the disorder. Either (1) or (2) may lead to a positive identification of a causative gene.

3. The construction of permanent cell lines expressing the cloned complete receptors. This will allow for in vitro automated screening of drugs with potential to interact with the GABA or brain nicotinic receptor. Thus, one could optimise molecular variations favouring delivery to brain sites and subtype specificity (as is needed for nicotinic reinforcement in Alzheimer's pathology and for the GABAA receptor in focal epilepsy, as examples). Such receptor-implanted cell lines will become a future staple of the pharmaceutical industry.