Expression of neuronal nicotinic receptors in Xenopus oocytes has shown that several different subunit combinations are functional, with a range of pharmacological and biophysical properties. In the nervous system, nicotinic receptors are found on the soma or the presynaptic terminals of neurones: the precise molecular identification of these receptor subtypes remains a challenge to pharmacology.
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INTRODUCTION FOR NICOTINE ADDICTION WORKSHOP BY MARCUS MUNAFO AND MIKE MURPHY: The ICRF General Practice Research Group in Oxford recently hosted a workshop on the theme of "Neuroscience, molecular genetics and nicotine addiction". It was chaired by Professor Neal Benowitz from the University of California at San Francisco, a specialist in the field of nicotine pharmacology spending an invited week in the University to work with the Group, and included about 30 other experts from groups around the UK and Sweden, including the ICRF Molecular Pharmacology Unit in Dundee and the ICRF Health Behaviour unit in London. Their backgrounds included neuroscience, molecular genetics, genetic epidemiology, pharmacology, psychology and clinical medicine, but they were united in a belief about the importance of tobacco control.
It is now recognised that addiction to nicotine lies at the heart of the tobacco control problem. Without the marketing of tobacco its use would dwindle. But while it is so widely promoted smoking research focuses heavily on factors affecting why people continue smoking after trying it and how to get them to stop with psychological support and the use of effective drugs. Although tobacco use is rightly viewed as a social and psychological problem we also need to see it as a pharmacological one with a potential genetic contribution to the addictive process. The need to integrate knowledge about the brain chemistry underlying addiction, the molecular genetics of these cellular processes, and how the drugs which are effective work, was the workshop's subject.
Each of the meeting's 4 sessions kicked off with a brief introduction by speakers who had precirculated draft papers. The sessions considered in turn: (1)how nicotine is metabolised and cleared from the body (2)the brain receptors in which nicotine "docks" to exert its effects (3)the brain pathways involving the neurotransmitter,dopamine,which are thought to contribute to the rewarding properties of nicotine which reinforce a smoker's desire to smoke (4)other non-dopamine pathways that are activated by nicotine and may underly such effects as nicotine withdrawal symptoms.
Understanding how the drugs which are effective (Nicotine Replacement Therapy and some antidepressants) interact with the expression of individual genotypes in the brain and elsewhere to predict success in quitting smoking will help to shed light on which bits of the brain are important in nicotine addiction at both the anatomical and cellular level. The workshop participants emerged pleased to have caught up with advances in fields which were not their speciality and with a strong sense that more enduring closer contact and cooperation will be possible in the future.
1. Approximately 50 years ago, the work of William Paton and Eleanor Zaimis in this country established the first clear distinction between the nicotinic receptors (nAChRs) at the neuromuscular junction of skeletal muscle and those on postganglionic autonomic neurones, namely their different sensitivity to decamethonium and hexamethonium (Paton & Zaimis, 1949). This distinction between muscle and neuronal nAChR remained essentially unchanged until molecular cloning revealed that the subunits, which are the building blocks for agonist-activated ion channels in general, and for neuronal nAChRs in particular, are bewildering diverse. The potential for molecular diversity in the neuronal nAChRs themselves is therefore very great, and is stretching to the limits our capacity to characterise receptors by their pharmacological and functional properties.
2. We now know quite a lot about the muscle nAChR; it may be useful to summarise these facts in order to provide a reference point for the related (but much less understood) neuronal nAChRs. In normal innervated muscle, nAChRs are a homogeneous population which is highly localised to the synaptic folds under the presynaptic terminal. Muscle nAChRs are pentamers of five subunits (Alpha-Beta-Gamma-Delta for foetal and denervated muscle, Alpha-Beta-Delta-Epsilon for adult muscle) arranged quasi-symmetrically around a central pore: the topology may be Alpha-Gamma-Alpha-Delta-Beta (or Alpha-Beta- Alpha-Delta-Gamma) (Karlin & Akabas, 1995; Unwin, 1998). At the arrival of a presynaptic action potential, the concentration of ACh in the synaptic cleft rises to millimolar levels for a very short time (0.2 milliseconds). Two molecules of ACh bind to the receptor, causing a conformational change that leads to the channel opening. The channel when open is permeable to cations, such as sodium, potassium and to a lesser extent calcium: in a normal neuromuscular synapse this leads to a depolarisation, which triggers with a high safety margin a postsynaptic action potential, and consequently muscle contraction. The channel opening is shortened at depolarised membrane potential (Edmonds et al., 1995). The properties of the open channel are determined by a stretch of amino acid residues which form the second transmembrane domain and line the open pore, whereas the binding sites for ACh are formed by the N-terminal extracellular domains of the Alpha subunits - it is debated whether other subunits such as Delta and Gamma participate to the site directly (Galzi & Changeux, 1994). Even if the two sites are made up exclusively of Alpha subunit residues, the shape of the two sites is different, because the two Alpha subunits find themselves surrounded by different subunits (Beta/Gamma or Gamma/Delta if we follow the first hypothetical topology) which affect them in a different way (Unwin, 1998). The consequence is that the two sites can be distinguished pharmacologically- albeit somewhat laboriously (Sine et al., 1990). Finally, prolonged application of ACh or a similar agonist - even at low levels - desensitises the receptor: at a normal synapse this is never allowed to happen by diffusion and enzymatic degradation of ACh (Edmonds et al., 1995).
3. Let us now consider the situation for neuronal nAChRs. First of all, neuronal nAChRs, native or recombinant, are also pentamers of five subunits: however, the number of neuronal nAChR subunits cloned so far has reached twelve (Alpha2-Alpha10 and Beta2-Beta4).
4. Which receptors are assembled out of these subunits? One way of looking at the problem is to check what the minimum subunit requirement is for the production of a functional receptor. This has been done by heterologous (over)expression of subunit combinations (mostly in the Xenopus oocyte system; for a review see McGehee & Role, 1995). In this system the following rules of assembly apply:
- a few Alpha subunits (only Alpha7, Alpha8 and Alpha9) can form a receptor when expressed alone.
- other Alpha subunits (Alpha2, Alpha3, Alpha4 and Alpha6) can only form a functional receptor if expressed with a Beta subunit (Beta2 or Beta4); these Alpha/Beta receptors have a stoichiometry of 2 Alpha to 3 Beta.
- two subunits (Alpha5 and Beta3) can form a receptor only if expressed together with an Alpha/Beta pair (i.e., Alpha2-Alpha4 plus Beta2 or Beta4; Ramirez-Latorre et al., 1996;Groot-Kormelink et al., 1998).
5. These minimal recombinant nAChR have been characterised in oocytes and - to a lesser extent - mammalian cell lines. In general, homomeric receptors, such as Alpha7, are very highly permeable to calcium (nearly as much as the NMDA-type of glutamate receptor, Seguela et al., 1993; Rogers & Dani, 1995) and desensitise very quickly in response to high agonist concentrations. These receptors are blocked by the peptide toxin alpha-bungarotoxin at nanomolar concentrations (though not irreversibly, as muscle type is).
6. Heteromeric receptors have a calcium permeability that is intermediate between those of Alpha7 and muscle nAChR. They are not blocked by alpha-bungarotoxin and their response decays more slowly upon continued agonist application (Gerzanich et al., 1998). Clearly these receptors have considerable scope for diversity of composition. Because of the lack of the classical, ideal tools for receptor characterisation, i.e., selective competitive antagonists, these receptors have been characterised on the basis of their agonist sensitivity. This method is far from ideal because it is not robust (for example, differences in the geometry of the preparation used or the method of application; see Sivilotti et al., 2000). Nevertheless, it is now established that, for example, Beta2-containing receptors (which may be more important in the central nervous system) are more sensitive to nicotine and DMPP than to cytisine. The order of potency is reversed for Beta4-containing receptors, which are particularly important in the autonomic nervous system (Luetje & Patrick, 1991; Covernton et al., 1994). The discovery (and increased availability) of a range of conotoxin peptides which act as competitive antagonists of nAChR offers the promise of more selective tools for investigation of heteromeric nAChR in the future.
7. All neuronal nAChRs display inward rectification, that is, they pass more current when the membrane is hyperpolarised: this phenomenon is much more marked than for muscle nAChR, as neuronal channels pass very little current at potentials more depolarised than -20 mV. This means that neuronal nAChR activation will be more effective when it does not coincide temporally with other excitatory events. This behaviour is the opposite of that of the NMDA receptor, which has been described as a coincidence detector.
8. Expression of nAChR subunits is widespread in the nervous system, but the subunits expressed depend upon the anatomical area and the developmental stage. The Alpha4 and Beta2 subunits appear to be predominant in the central nervous system whereas Alpha3 and Beta4 are more important in the peripheral nervous system. Examining the properties of native nAChRs and comparing them to those of recombinant receptors has allowed the identification of broad classes of native receptors (McGehee & Role, 1995). This identification has been essentially confirmed by the effects of functional deletion of specific subunits in transgenic mice (Cordero-Erausquin et al., 2000). Thus, different types of hippocampal neurones display responses to ACh application that differ in kinetics and antagonist sensitivity (Albuquerque et al., 1997). This strongly suggests the presence of Alpha7-type receptors on the somata of some interneurones (i.e., CA1 stratum radiatum): these receptors can mediate fast synaptic activity, although the importance of the contribution of nicotinic signal to the total excitatory drive of these and other central neurones is not known (Frazier et al., 1998a,b; Hefft et al., 1999). Other hippocampal neurones, for instance, dentate gyrus interneurones, display slower somatic nicotinic responses, which are likely to be non-Alpha7. Because each interneurone synapses onto a large number of principal cells, the sphere of influence of nAChRs is increased by their location on interneurones (Jones et al., 1999). Neuronal nAChRs have long been known to be essential to the fast synaptic excitation of spinal cord interneurones (Renshaw cells) and autonomic ganglion neurones (Brown, 2000).
9. Another important location of nAChRs is on axons or presynaptic terminals: depending on the anatomical area, activation of these receptors can increase the release of glutamate, GABA, dopamine or noradrenaline. Activation of these nAChRs can therefore exert extensive modulatory effect on other neurotransmitter systems (McGehee et al., 1995). Even though direct characterisation of these nAChRs is difficult because of their inaccessibility, it is clear that different areas have different presynaptic receptors and that these mostly belong to the non-Alpha7 group; on autonomic neurones, somatic receptors are known to be different from presynaptic nAChR. We still don't know whether these presynaptic receptors are activated physiologically, i.e., whether they see neurotransmitter, or how the different receptor types are targeted to different cellular domains (i.e., presynaptic terminal vs. cell body; Wonnacott, 1997).
10. Within this broad consensus on nAChR diversity, problems of interpretations still persist, particularly with respect to the extrapolation of data from recombinant nAChR, and therefore to the identification of the precise subunit composition of native nAChRs. Even if recombinant receptors are a good approximation of native ones (and there is evidence for caution, see Sivilotti et al., 1997; Lewis et al., 1997), the starting questions were different: in oocytes we searched for the minimal receptor compositions that are functional, whereas in the nervous system we started from knowing which subunits are expressed (usually several in each neurone!) and wanted to know which receptors result from these subunits. Antibody and antisense work shows that native neuronal nAChRs do not have the minimal composition that can easily be studied in oocytes. For instance, the main synaptic receptor of autonomic ganglia may have a composition of Alpha3Beta4Alpha5 +Beta2 (Vernallis et al., 1993). Characterising this receptor in an oocyte is difficult, because the expression of several subunits leads to the production of a mosaic of receptors, among which isolating and characterising the Alpha3Beta4Alpha5Beta2 receptor would be impossible, as even expressing just three subunits, such as Alpha3Beta4Alpha5 produces two receptor populations (Alpha3Beta4 and Alpha3Beta4Alpha5). Other examples indicate that some native Alpha7-containing receptors are not homomers, but contain other neuronal subunits and differ in kinetic properties (and possibly alpha- bungarotoxin sensitivity) from the homomers we can study in oocytes (Yu & Role, 1998; Cuevas & Berg, 1998; Cuevas et al., 2000).
11. In other words - even if we know what the effects of a subunit are in a simple, minimal-composition receptor - it is difficult to predict how the presence or absence of this subunit is going to affect a real, complex receptor. The last example shows that we cannot rely on Alpha7 to always manifest itself by fast kinetics and high alpha-bungarotoxin sensitivity. Similarly (even in oocytes), adding Alpha5 to the subunits expressed does not have a consistent effect on ACh sensitivity (Gerzanich et al., 1998). ACh potency can be greatly decreased by Alpha5 (Alpha4Beta2Alpha5 receptors vs. Alpha4Beta2 receptors; Ramirez-Latorre et al., 1996) or can be left unchanged (Alpha3Beta4Alpha5 vs Alpha3Beta4 receptors; Wang et al., 1996).
12. Thus, advances in molecular biology and electrophysiology of neuronal nAChRs reveal a complex picture, which still poses the challenge of elucidating the precise molecular composition and actual physiological role of these receptors.
In conjunction with the Report from Royal College of Physicians.
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