EARLY PHARMACOLOGICAL STUDIES AND THE IMPACT OF MOLECULAR GENETIC TECHNIQUES
Traditionally receptors have been classified according to their pharmacology. Each neurotransmitter acts on its own family of receptors and these receptors show a high degree of specificity for their transmitter. Thus, the receptors on which acetylcholine (ACh) works do not respond to glutamate (or any other neurotransmitter) and vice versa. Diversity of neurotransmitter action is provided by the presence of multiple receptor subtypes for each neurotransmitter, all of which still remain specific to that neurotransmitter. This principle is illustrated by the simple observations outlined in
Neurotransmitters, Drugs and Brain Function. Edited by R. A. Webster ©2001 John Wiley & Sons Ltd
Chapter 1 which showed that since muscarine mimicked some of the actions of ACh (but not all) while nicotine mimicked the other actions of ACh, then ACh probably acted on two distinct types of receptors. The fact that atropine antagonised the muscarinic effects of ACh but not the nicotinic effects, while tubocurarine blocked the nicotinic effects provided firm evidence for this concept. These simple qualitative observations by Langley and others at the beginning of the twentieth century led to the development of more quantitative pharmacological methods that were subsequently used to identify and classify receptors. These methods were based on the use of both (1) agonist and (2) antagonist drugs:
(1) If a series of related chemicals, say noradrenaline, adrenaline, methyladrenaline and isoprenaline, are studied on a range of test responses (e.g. blood pressure, heart rate, pupil size, intestinal motility, etc.) and retain exactly the same order of potency in each test system, then it is likely that there is only one type of receptor for all four of these catecholamines. On the other hand, if, as Ahlquist first found in the 1940s, these compounds give a distinct order of potency in some of the tests, but the reverse (or just a different) order in others, then there must be more than one type of receptor for these agonists. (3) If one set of these responses can be blocked (antagonised) by a drug that does not affect the other responses (e.g. propranolol blocks the increase in heart rate produced by adrenaline, but not the dilation of the pupil evoked by adrenaline) then this is good evidence that adrenoceptors in the pupil are not the same as those in the heart.
In fact, careful quantitative analysis of the order of activity of the agonists in each test, and of the precise potency of antagonists (see Chapter 5 for quantitative detail) has often successfully indicated, although rarely proved, the presence of subclasses of a receptor type (e.g. different muscarinic receptors). The affinity of receptors for selective antagonists determined using the Schild method was a mainstay of receptor classification throughout the second half of the twentieth century. Thus, a muscarinic receptor can be defined as a receptor with an affinity for atropine of around 1 nM and the Ml subtype of muscarinic receptor can be identified as having an affinity of around 10 nM for the selective antagonist, pirenzepine while muscarinic receptors in the heart (M2 subtype) are much less sensitive to pirenzepine block (KB ~ 10~7 M).
Classification of receptors according to agonist potency can be problematic because agonist potency depends partly on the density of receptors in the tissue and therefore use of selective antagonists has become a mainstay of receptor identification and classification. The development of radioligand binding techniques (see Chapter 5 for principles) provided for the first time a means to measure the density of receptors in a tissue in addition to providing a measure of the affinity of drugs for a receptor and allowed the relative proportion of different receptors in a tissue to be estimated.
These approaches to receptor identification and classification were, of course, pioneered by studies with peripheral systems and isolated tissues. They are more difficult to apply to the CNS, especially in in vivo experiments, where responses depend on a complex set of interacting systems and the actual drug concentration at the receptors of interest is rarely known. However, the development of in vitro preparations (acute brain slices, 'organotypic' brain slice cultures, tissue-cultured neurons and acutely dissociated neuronal and glial cell preparations) has allowed more quantitative pharmacological techniques to be applied to the action of drugs at neurotransmitter receptors while the development of new recording methods such as patch-clamp recording has allowed the study of drug action at central neurons to be made at ever more detailed levels.
Today we know not only that there is more than one type of receptor for each neurotransmitter, but we also know a great deal about the structural basis for the differences between receptor subtypes which are due to differences in the amino-acid sequence of the proteins which make up the receptor. How do we know this?
Finding the amino-acid sequence of a receptor protein has been approached in three main ways. The final aim of all three methods is to obtain a cDNA clone coding for the protein since the base sequence of this DNA allows the amino-acid sequence of the protein to be predicted:
(1) From purified receptor protein, obtain partial amino-acid sequence information which will allow molecular biologists to isolate the gene (or genes) coding for the receptor.
(2) cDNA library screening. From a receptor-rich tissue, isolate mRNA and create from this, a cDNA library. The library is then screened by, for example, functional expression in Xenopus oocytes or mammalian cell lines, for the proteins coded by the library. If positive expression is obtained, the library is subdivided until a single cDNA clone for the receptor is isolated.
(3) Homology screening. Using oligonucleotide probes based on known receptor sequences, search cDNA libraries for homologous sequences which may code for related receptors. The clones are then isolated and sequenced and used in expression studies to confirm the identity of the receptor.
The first tentative steps towards determining the structure of individual receptors were taken by protein chemists. A high-affinity ligand that binds specifically to the receptor (generally an antagonist) was identified by traditional pharmacological methods and attached to the matrix of an appropriate chromatography column. A tissue source, rich in receptors, is homogenised and the cell membranes disrupted with detergents to bring the membrane bound proteins into solution. This solution is then passed through the affinity column and the receptor of interest will stick to the column hence separating it from all the other proteins in the tissue. The receptor is then eluted from the column using a solution of ligand specific for the receptor. This strategy allowed isolation of the nicotinic acetylcholine receptor from the electric organ of the Californian ray (Torpedo). Almost 40% of the protein content of this tissue is ACh receptor. The isolation method used a snake toxin from the venom of the Taiwan banded krait (a-bungarotoxin) as the ligand of the affinity column and the purified receptor was eluted from the column using a high concentration of the competitive antagonist, tubocurarine. Following isolation of the protein, a partial N-terminus amino-acid sequence was obtained and from this sequence, oligonucleotide probes were made which were then used to screen a cDNA library to isolate a clone for the receptor. Since DNA sequencing is much faster than protein sequencing, the DNA sequence of the clone is then used to provide the amino-acid sequence of the receptor.
It is often valuable to classify receptors according to their mechanism of action, because this is intimately related to structure. The neurotransmitter receptors in the brain are of two main types classified according to their structure and mechanism of action:
(1) Ion channel receptors
(2) G-protein-coupled receptors
The ion channel receptors are relatively simple in functional terms because the primary response to receptor activation is generated by the ion channel which is an integral part of the protein. Therefore, no accessory proteins are needed to observe the response to nicotinic AChR activation and the full functioning of the receptor can be observed by isolating and purifying the protein biochemically and reconstituting the protein in an artificial lipid membrane. In contrast, the G-protein-coupled receptors require both G-proteins and those elements such as phospholipase-C illustrated in Fig. 3.1, in order to observe the response to receptor activation (in this case a rise in intracellular calcium concentration resulting from the action of IP3 on intracellular calcium stores).
Most receptors function as mediators of synaptic transmission between neurons. Figure 3.1 illustrates this for the case of a generic glutamatergic synapse. At this synapse glutamate is released from the presynaptic nerve terminal and acts on two different types of fast ionotropic glutamate receptors embedded in the postsynaptic membrane: AMPA receptors mediate an extremely rapid (within 1 ms) response to glutamate release resulting in a rapid depolarisation of the postsynaptic membrane (EPSP). On a slower time scale, the NMDA receptors mediate a slower EPSP which lasts over 100 ms, is carried partly by calcium ions and is voltage-dependent due to blocking of the NMDA receptor channel by Mg ions at negative membrane potentials. The AMPA receptor provides the depolarisation necessary to relieve the Mg block of the NMDA channel and so the calcium influx through the NMDA channel in effect provides a means to integrate synaptic activity mediated by the fast AMPA receptors. The synapse shown in Fig. 3.1 also illustrates that G-protein-coupled glutamate receptors may be located at the synapse, or perisynaptically and therefore can mediate slow synaptic transmission (on a time scale of 100 ms to seconds) whose characteristics will depend on the particular G-protein which is coupled to the metabotropic receptor. In this case, the receptor is coupled by Gq to phospholipase-C and results in IP3 and diacyl-glycerol (DAG) production which in turn regulate intracellular calcium concentration and protein kinase-C activity. Thus, at any glutamatergic synapse in the brain there is the potential for a single neurotransmitter to generate fast and slow signals with particular characteristics which depend on the properties of the neurotransmitter receptors expressed in the target cell membrane.
The definitive classification of receptors is by amino-acid sequence analysis. Since all properties of the receptor are determined by the amino-acid sequence of the protein this method has the final say. The explosion in use of molecular genetic techniques in the final decade of the twentieth century has led to the cloning and sequencing of the genes of all the known neurotransmitter receptors in the brain. From the gene sequence, the amino-acid sequence of the receptor protein can be inferred and hence a final classification of all receptors can be made. Ultimately, the human genome sequencing programme will mean that the amino-acid sequence of all human receptors will be known. Does this mean pharmacologists can now retire happy in the knowledge that all is now known that there is to know? Far from it! Gene cloning and sequencing has unveiled an increasingly vast diversity among receptor types which could barely have
Presynaptic terminal mGtuR
(j-protein Phoapholipase C
Figure 3.1 Schematic representation of a generic excitatory synapse in the brain. The presynaptic terminal releases the transmitter glutamate by fusion of transmitter vesicles with the nerve terminal membrane. Glutamate diffuses rapidly across the synaptic cleft to bind to and activate AMPA and NMDA receptors. In addition, glutamate may bind to metabotropic G-protein-coupled glutamate receptors located perisynaptically to cause initiation of intracellular signalling via the G-protein, Gq, to activate the enzyme phospholipase and hence produce inositol triphosphate (IP3) which can release Ca2+ from intracellular calcium stores been imagined by pharmacologists only 20 years ago. The properties and subtle functional differences between receptor subtypes can be studied in increasing detail utilising receptor expression systems such as Xenopus oocytes and clonal mammalian cell lines where single receptor populations at high density can be studied without the complications arising from the diversity of receptors present in brain tissue, or the difficulty of recording responses from receptors in the brain. Pharmacology has now entered the era of 'post-genomic' research in which the challenge is to utilise the diversity of receptor types revealed to us by gene cloning techniques in the development of subtype selective drugs. The hope is that if this diversity of receptor subtypes is matched by diversity of function in the brain, then subtype-selective drugs may provide the means to selective therapeutic agents with a minimum of side-effects for use in treating diseases of the brain.
The following sections of this chapter will consider some general and comparative aspects of receptor structure and function. More detailed material on these topics may be found in the relevant chapters on individual neurotransmitters.
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