Modulation of neuronal nicotinic AChRs by ethanol
The data presented here suggest that different neuronal nicotinic receptor subtypes can differ in their characteristics of modulation by physiologically encountered concentrations of ethanol. Under the conditions employed in these experiments, the different subtypes appeared to fall into three categories: (i) α3β4-type effects are characterized by the presence of both potentiation and inhibition at low ethanol concentrations in some cells, and robust potentiation at high ethanol concentrations; (ii) generally α3β2, α4–1β2 and α4–1β4 receptors are less sensitive at low concentrations, but show potentiation at high EtOH concentrations; (iii) α7-type receptors are relatively insensitive at low EtOH concentrations, but show both potentiation and inhibition at high EtOH concentrations. However, this characterization may be somewhat arbitrary. The low concentration effects observed with α3β4 were relatively rare events. Therefore, it is possible that under other conditions, such as in the presence of Ca2+ (Dildy-Mayfield & Harris, 1995), the other subtypes may show greater sensitivity than they have in this particular set of experiments.
In contrast to the variability of the effects of low ethanol concentrations, potentiation of responses at high ethanol concentrations was nearly always seen. However, Figure 5 and Table 1 demonstrate that the average magnitude of the potentiation produced by 300 mM ethanol was different for different combinations. The order of decreasing sensitivity to ethanol was α3β4>α4β2>α3β2>α4β4>α7. The responsiveness of the α3β4 combination was considerably greater than that of any other. This cannot be attributed to the presence of an independent site on the β4subunit, since the α4β4 combination showed the least tendency to potentiation of any of the heteromeric combinations. Nor can it be attributed solely to the presence of the a3 subunit, as the α3β2 combination showed less potentiation than the α4β2 combination. It would seem that both α3 and β4 subunits are required. Perhaps both subunits contribute juxtaposed domains which together form a binding site for alcohol. In other combinations, the domains may be less well aligned and the synergism would not occur.
Studies on the responses of nicotinic AChRs in ganglia, which also express α3 and β4 subunits, show that the activity of the receptors can be both upregulated and downregulated (Valenta et al., 1993; Gurantz et al., 1994). We also see this with α3β4 receptors expressed from cDNAs in oocytes. The variability of the degree of modulation of α3β4 responses by low concentrations of ethanol has several interesting implications. If ethanol worked simply by acting on a hydrophobic pocket in the protein or on the annular lipid to disrupt protein structure, then we would perhaps expect more consistency in the degree and direction of current modulation. The inconsistency at low concentrations tends to argue against a simple change in membrane fluidity as the sole mechanism. Also, studies on the Torpedo receptor in lipid vesicles suggest that the lipid-protein interface, at least deep in the bilayer, is quite insensitive to the presence of ethanol up to 0.9 M (Abadji et al., 1994). One possibility is that the rapid potentiation and inhibition of nicotinic responses at low concentrations of ethanol may be mediated by intracellular signalling pathways. This may involve intimate association of the receptor with a regulatory protein, such as occurs between the N-methyl-D-aspar-tate (NMDA) receptor and protein tyrosine kinase Src (Yu et al., 1997).
The α3 and β4 subunits are not just restricted to ganglia, but are also found throughout the brain (Dinely-Miller & Patrick, 1992). α3 and β4 subunits also occur together in receptors in the mammalian nervous system (Flores et al., 1996). In oocytes, this combination is characterized by a long open time (Papke & Heinemann, 1991) and like all neuronal nicotinic receptors, is more permeable to Ca2+ than muscle AChRs (Mulle et al., 1992; Vernino et al., 1992; 1994). Nicotinic single channel activity with analogous characteristics has been observed in the medial habenula (Mulle et al., 1992; Connolly et al., 1995), where α3 and β4 subunits are also expressed (Duvoisin et al., 1989), and so it seems probable that the observations described here will have relevance to the actions of ethanol on native nicotinic AChRs containng a3 and β4 sub-units in brain tissue. Both these subunits are also expressed in PC12 cells (Boulter et al., 1990), where Nagata et al. (1996) have recently shown that low concentrations of ethanol (30 μM to 10 mM) can produce variable effects of potentiation and inhibition of nicotinic responses.
The effects of ethanol on the rat α7 homomeric receptor presented here appear to contradict those obtained for the chick α7 in another study. In the work of Yu et al. (1996) ethanol caused a dose-dependent inhibition of the nicotine-induced current response (IC50 = 33 mM), whereas we observed mixed inhibition/potentiation at concentrations above 30 mM. The reasons for this difference are unclear, as the agonist concentrations used were similar (10 μM vs 10–30 μM nicotine) and acute ethanol applications were used (although not using a ‘one-shot’ protocol). Also, the use of higher agonist concentrations would not seem to explain the differences. In one cell, the response of rat α7 to 100 μM nicotine was only reduced by 5% in the presence of 100 mM ethanol. A second response in the same cell to 300 μM nicotine was reduced by only 2.5%. In a second cell the response to 300 μM nicotine was not altered in the presence of 300 mM ethanol (data not shown). It would therefore seem possible that there is a species difference between the rat and chick α7 receptors in their pattern of ethanol modulation. Several pharmacological differences between these two receptors have previously been described - for instance the agonist l,l-dimethyl-4-phenylpi-perazinium iodide (DMPP) is a potent near-full agonist on the rat α7 receptor, but a very weak partial agonist on the chick α7 receptor (Bertrand et al., 1992; Seguela et al., 1993), even though there is considerable (90%) amino acid sequence homology between them (Couturier et al., 1990; Seguela et al., 1993). However, DeFiebre et al. (1995) suggested that such inhibition by EtOH may also occur with the rat α7 receptor.
The suggestion that the observations described here may have importance beyond the oocyte is reinforced by the fact that mecamylamine, a nicotinic antagonist, can antagonize the mesolimbic dopamine-activating properties of ethanol (Blomqvist et al., 1993). Therefore, it seems possible that alcohol enhancement of nicotinic receptor activity in the mesolimbic pathway may contribute the mutual reinforcement of drinking and smoking behaviour. Similarly, it is possible that the modulation of neuronal nicotinic receptor subtypes may contribute to the induction of alcohol-dependence due to chronic high alcohol exposure. The concentration of ethanol at which the potentiating effect starts to occur in all the hetero-meric receptors tested here (and sometimes with the α7 receptor) is equivalent to that regularly experienced by heavy drinkers (i.e. <30 mM, which is equivalent to approx. 10 units; 1 unit = 8 g=10ml EtOH).
In rat substantia nigra reticulata and ventral palladium, Criswell et al. (1993) examined the nicotine-induced changes in neuronal firing rate. In 7 of 9 cells which were excited by nicotine, the increase in firing rate was enhanced by alcohol. However, Frölich et al. (1994) noted that alcohol could inhibit the excitatory effects of nicotine, kainate and NMDA on neuronal firing rates in rat locus coereleus, where α3β4 is also expressed. From these results it is apparent that the effects of ethanol on nicotinic receptors are not uniform in the brain, but may be subtype specific, or depend upon the intracellular signalling status of the cell under investigation.
Criswell and colleagues also noted that the degree of inhibition of the NMDA response declined with prolonged incubation with ethanol. We have observed similar evidence of an initial strong sensitivity to low concentrations of ethanol, which fades with repeated exposure. This raises the possibility that if an individual rapidly consumes even a small amount of alcohol, then there could be a window of time before tolerance sets in during which the person's performance is strongly affected. For a short period, driving ability could be compromised even though the blood alcohol level is well below the legal limit.
Another implication of these results is that in the brain, the control of the activity of nicotinic AChRs may be highly dynamic and specific for each subunit combination. The diversity of the Mill - MIV cytoplasmic domains may mediate some of this plasticity, enabling each receptor subtype to respond rapidly to a particular set of intracellular signalling influences. Unfortunately, dialysis of neurones during patch clamp may remove or disrupt some of these signalling pathways.
In the light of the present results, it is tempting to suggest that the direct effects of alcohol on neuronal nicotinic receptors is responsible for the intense need to smoke that some people feel when drinking alcohol. It will be informative to determine whether it is the inhibitory effect or the potentiating effect which is important, and again which subtypes are involved. The answers to these questions will help define the mechanisms of reward in drug dependence. They may also help in the design of more effective strategies for smoking cessation, for if the effects described here occur in the brain, then there may be a case for advising those hoping to give up smoking that they should avoid heavy drinking at all costs!.