Cellular approaches to the interaction between cannabinoid receptor ligands and nicotinic acetylcholine receptors

European Journal of Pharmacology 731 (2014) 100–105

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Cellular approaches to the interaction between cannabinoid receptor ligands and nicotinic acetylcholine receptors Murat Oz a,n, Lina Al Kury a, Susan Yang Keun-Hang b, Mohamed Mahgoub a, Sehamuddin Galadari c a Functional Lipidomics Branch, Department of Pharmacology, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain, Abu Dhabi, UAE b Department of Biological Sciences, Schmid College of Science and Technology, Chapman University, 1 University Drive, Orange, CA 92866, USA c Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, Al Ain, UAE

art ic l e i nf o

a b s t r a c t

Article history: Received 27 November 2013 Received in revised form 24 February 2014 Accepted 10 March 2014 Available online 16 March 2014

Cannabinoids are among the earliest known drugs to humanity. Cannabis plant contains various phytochemicals that bind to cannabinoid receptors. In addition, synthetic and endogenously produced cannabinoids (endocannabinoids) constitute other classes of cannabinoid receptor ligands. Although many pharmacological effects of these cannabinoids are mediated by the activation of cannabinoid receptors, recent studies indicate that cannabinoids also modulate the functions of various integral membrane proteins including ion channels, receptors, neurotransmitter transporters, and enzymes by mechanism(s) not involving the activation of known cannabinoid receptors. Currently, the mechanisms of these effects were not fully understood. However, it is likely that direct actions of cannabinoids are closely linked to their lipophilic structures. This report will focus on the actions of cannabinoids on nicotinic acetylcholine receptors and will examine the results of recent studies in this field. In addition some mechanistic approaches will be provided. The results discussed in this review indicate that, besides cannabinoid receptors, further molecular targets for cannabinoids exist and that these targets may represent important novel sites to alter neuronal excitability. & 2014 Elsevier B.V. All rights reserved.

Keywords: Cannabinoids Endocannabinoids Nicotinic acetylcholine receptors

1. Introduction Cannabis and tobacco are the most widely used plant based drugs of abuse throughout the world. Although cannabinoids and tobacco-based products have been used for medicinal, recreational, and ritual purposes for millennia, their pharmacological mechanisms are elucidated in the last century. The psychoactive components of these plants were identified as Δ9-tetrahydrocannabinol (THC) and nicotine, respectively. Most of the pharmacological actions of tobacco products are mediated by the nicotinic acetylcholine receptors, and the actions of CBs, on the other hand, appear to involve a more complex system consisting of endocannabinoid system and also various CB receptor ligands. Currently, CBs, according to their source, have been classified into three groups, mainly phytocannabinoids synthetic CBs, and endocannabinoids. The cannabinoid system is made up of CB receptors, their endogenous and exogenous ligands and the enzymes, substrates and precursors

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involved in their synthesis, release and degradation (for a recent review, see Pertwee et al. (2010)). Nicotinic cholinergic system, on the other hand, is made of nicotinic receptor ligands, synthesizing and degrading enzymes, and nicotinic acetylcholine receptors. The nicotinic receptors are a heterogeneous class of cationic channels that are widely distributed in the nervous system and skeletal muscle. They consist of homologous subunits encoded by a large multigene family, and their opening is physiologically controlled by acetylcholine or exogenous ligands such as nicotine (Hurst et al., 2013). Nicotine activation of these receptors causes, in addition to well-known postsynaptic depolarizing actions, a cascade of events by releasing several neurotransmitters that trigger various neuronal responses such as GABA and glutamate release, and regulate nicotine induced responses in various brain regions (Albuquerque et al., 2009; Hurst et al., 2013; Picciotto and Mineur, 2013). Interestingly, converging evidence indicates that cannabinoid and nicotinic systems interact with each other at cellular and neuronal network levels (for reviews, see Castane et al. (2005); Muldoon et al. (2013)). Both these compounds have addiction liability, and a high percent of comorbidity (Pertwee et al., 2010; Muldoon et al., 2013). CB receptors and nicotinic acetylcholine

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receptors overlap in many brain regions (Morales et al., 2008), such as the hippocampus and the mesolimbic dopamine system (for reviews, see Pertwee et al. (2010), Muldoon et al. (2013), and Cea-del Rio et al. (2012)). In several recent in vivo and behavioral investigations, influences of CB receptor activation on the nicotine-induced behavioral responses and the mechanisms of interaction between CBs and nicotinic receptors have been studied in detail (for a review, see Muldoon et al. (2013)). Cannabinoid receptor agonists have been shown to modulate the release and the turnover of acetylcholine in various brain areas involved in nicotine-induced behavioral responses (Tripathi et al., 1987; Gessa et al., 1998; Acquas et al., 2000). Similarly, memory-related effects of nicotine in the mouse elevated plus maze experiments were significantly suppressed by WIN, 55-212-2 a synthetic cannabinoid receptor agonist (Biala and Kruk, 2008). Collectively above mentioned studies suggest that cannabinoids can modulate nicotine-induced responses. In this report, we will focus on studies investigating the mechanisms of interaction between cannabinoid receptor ligands, especially endocannabinoids, and nicotinic receptors.

2. Direct interaction of cannabinoids with nicotinic receptors: Subsequent to the discovery of N-arachidonylethanolamide (anandamide; AEA), it was demonstrated that some of the pharmacological effects of AEA were not sensitive to CB receptor antagonists such as SR141716A (for a review, see Di Marzo et al. (2000); Wiley and Martin, (2002)). AEA still exerted cannabimimetic-like activities in the tetrad test and other behavioral tests carried out in CB1 receptor knock-out mice (Di Marzo et al., 2000; Baskfield et al., 2004). In addition, intrathecal injection of a selective CB1 antagonist could not completely inhibit the analgesic effects induced by THC and HU 210 (a synthetic cannabinoid structurally similar to THC), in spinal tail-flick reflex test in mice (Welch et al., 1998). THC and AEA-induced analgesia in the tail flick reflex test (for THC) and hot-plate test (for AEA) remained intact in CB1 knock-out (Zimmer et al., 1999) and in CB1 and CB2 knock-out mice (Racz et al., 2008). Furthermore, AEA has been shown to stimulate GTP-γ-S binding in brain membranes isolated from mice lacking CB1 receptors, and this effect was not altered by CB1 and CB2 antagonists (Di Marzo et al., 2002). In addition, recent studies investigating behavioral actions of cannabinoids also reported significant differences between THC and endocannabinoids in discriminative stimulus effects (Jarbe et al., 2003), and pointed out the possibility of receptor-independent actions of endocannabinoids effects. In fact, at the same time as the discovery of AEA, interaction of AEA and non-cannabinoid receptor targets such as L- type voltage gated Ca2 þ channels was reported in radioligand binding studies on the cortical (Johnson et al., 1993) and later in skeletal muscle membranes (Oz et al., 2000) suggesting that AEA is not selective for CB receptors. In recent years, several studies indicate that, in addition to activation of CB receptors, cannabinoids, particularly the endocannabinoids have direct, receptor-independent, and lipid-bilayer mediated actions on various membrane proteins, enzymes, and transporters (for reviews, see Oz (2006) and Pertwee et al. (2010)). Direct actions of endocannabinoids on nicotinic acetylcholine receptors were first reported in Xenopus oocytes expressing α7-nicotinic receptors (Oz et al., 2003, 2004). AEA and 2-AG noncompetitively inhibited α7-nicotinic receptor-mediated currents with IC50 values of 229 and 168 nM, respectively. AEA, at concentration range of 30–300 μM, did not inhibit specific [3H] nicotine binding in human frontal cortex (Lagalwar et al., 1999) and rat thalamic membranes (Butt et al., 2008), further suggesting a noncompetitive action of AEA on nicotinic receptors. This study

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also indicated that inhibition by AEA was not mediated by CB1 or CB2 receptors, since neither SR 141716A nor SR 144528 affected AEA inhibition of α7-nicotinic receptors (Oz et al., 2003). In addition, the effect of AEA was not sensitive to either pertussis toxin (PTX) treatment or to membrane-permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in AEA metabolism were ineffective in preventing the effect of AEA on nicotinic responses, suggesting that AEA itself acts on nicotinic receptors (Oz et al., 2003). In line with earlier findings, arachidonic acid (AA) (Vijayaraghavan et al., 1995), but not ethanolamine or glycerol, inhibited the function of α7-nicotinic receptors in a noncompetitive manner. The IC50 value (1 μM) for AA was significantly higher than those for 2-AG and AEA, suggesting that it was the intact endocannabinoid and not the metabolite AA that altered the function of α7-nicotinic receptor. Recently, Baranowska et al. demonstrated for the first time that direct inhibition of α7-nicotinic receptor function by endocannabinoids occurs under in vivo conditions that allow the separation of both cannabinoid receptor dependent and independent actions of endocannabinoids (Baranowska et al., 2008). In urethane anesthetized rats, methanandamide (metAEA) produced AM261insensitive inhibition of nicotine-induced tachycardiac responses mediated by the activation of α7-nicotinic receptors on the cardiac postganglionic sympathetic neurons. Similarly, results of nicotine withdrawal studies have shown that endocannabinoids modulate the somatic signs of nicotine withdrawal through non-CB1 receptors (Merritt et al., 2008). In this study, it was suggested that increased endocannabinoid levels were associated with enhanced nicotine withdrawal (Merritt et al., 2008) by a mechanism that does not involve CB1 receptors (Muldoon et al., 2013). In another study in urethane anesthetized rats, nicotine-induced activity of ventral tegmental area (VTA) dopaminergic neurons was suppressed by URB597, an inhibitor of FAAH, an enzyme that catabolizes AEA suggesting that increased AEA levels can regulate nicotinic receptors (Melis et al., 2008). Importantly the effect of URB597 on nicotine-induced responses was sensitive CB1 receptor antagonists and not mimicked by metAEA. Furthermore, based on sensitivity of nicotine-induced responses to genistein and other ligands, peroxisome proliferator activated receptor-α receptors have been proposed to mediate the actions of AEA on nicotinic receptors (Melis et al., 2008). However, tyrosine kinase modulators such as genistein used in this study has been shown to be positive allosteric modulators of nicotinic receptors (Gronlien et al., 2007, 2010) and modulate the activity of other ligand-gated ion channels (Dunne et al., 1998; Zhu et al., 2003; Huang et al., 2010) in a phosphorylation-independent manner (Hwang et al., 2003). Therefore further studies are needed to elucidate mechanisms of endocannabinoid actions on the nicotinic receptors of VTA neurons. Synthetic CBs, on the other hand, showed variations in their effects. For example, in Xenopus oocytes, WIN55,212-2 (10 mM) did not show any effect, but CP55940 inhibited α7-nicotinic receptor with an IC50 value of 2.7 mM (Oz et al., 2004). On the other hand patchclamp studies in cultured rat trigeminal ganglion neurons indicated that native nicotinic receptor was inhibited by WIN55,212-2 with an IC50 value about 3 mM (Lu et al., 2011). The effect of WIN55,212-2 was not sensitive to CB receptor antagonists and GDP-β-S treatments and therefore it was concluded that WIN55,212-2 directly acted on nicotinic receptors (Lu et al., 2011). It is noteworthy to mention that synthetic CBs have rather diverse chemical structures. Due to this structural diversity, it is not surprising that their effects vary significantly depending on the chemical structure and cell type tested Oz et al., 2004; Lu et al., 2011. Among phytocannabinoids, THC and cannabinol, up to 10 μM had no effect on α7-nicotinic receptor function (Oz et al., 2004; Mahgoub et al., 2013). However, cannabidiol inhibited α7-nicotinic receptor with an IC50 value of 11.3 mM (Mahgoub et al., 2013).

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The effect of cannabidiol, like endocannabinoids, was noncompetitive and voltage- independent. Although the importance of cannabidiol actions in overall pharmacological effects of cannabis is not clear, it is noteworthy to mention that cannabidiol has a terpenoid structure and there are over 120 terpenes (terpenoid fragrance molecules) manufactured within the cannabis plant. Our recent studies indicated that many of these terpenes also modulate functional properties of several ion channels and receptors including nicotinic receptors (Ashoor et al., 2013a,b). Although THC alone mediates most of the psychoactive effects of cannabis, some of the recent studies suggest that other phytochemicals found in the cannabis plant also contribute to its overall effects (Russo and McPartland, 2003; Russo, 2011). Therefore, it is likely that different terpene-based chemicals found in cannabis plant would also modulate a repertoire of ion channels and contribute to the pharmacological actions of cannabis. Pharmacological importance of CB actions on nicotinic receptors is currently unknown. However, based on the current knowledge on nicotinic receptor pharmacology in the nervous system, we can infer some assumptions. It is important to note that, in the nervous system, most of α7-nicotinic receptors are located at the presynaptic buttons of GABAergic and glutamatergic neurons. Neuronal nicotinic receptors, specifically the α7-nicotinic receptors, have high Ca2 þ permeability to the extent that Ca2 þ influx through these channels at presynaptic terminals may cause transmitter release and modulate both excitatory and inhibitory synaptic neurotransmission in various cortical areas (Albuquerque et al., 2009;Hurst et al., 2013). In this context, it is likely that CBs can directly interact with both pre- and post-synaptically located nicotinic receptors and modulate both glutamatergic and GABAegic synaptic transmission in various brain regions. For example, in an in vitro study, in presence of the CB1 receptor antagonist SR141716A, both the amplitude and the frequency of choline (α7-nicotinic receptor agonist)-induced increase of the spontaneous inhibitory postsynaptic currents were significantly decreased by 1 μM AEA (Oz et al., 2005), suggesting that α7nicotinic receptor modulation of GABAergic neurotransmission is altered by AEA. Similarly, α4β2-nicotinic receptors located in presynaptic terminals (synaptosomes) of hypothalamic neurons are functionally inhibited by AEA (Butt et al., 2008). In conclusion, the ability of nicotinic receptors to increase neurotransmitter release is likely to underlie at least some of its effects on cholinergic pharmacology and CBs can be expected to modulate these behavioral and cellular responses by acting directly on the nicotinic receptors. Pre- and post-synaptic actions of nicotinic receptors in hippocampus and brain regions suggest that modulation of the function of these receptors can play roles under several physiological and pathological conditions such as reward circuits, appetite, stress-related behaviors, neuronal development, and neurodegenerative disorders (Picciotto and Mineur, 2013;Oz et al., 2013). Future studies will further provide more evidence for the direct modulation of presynaptic nicotinic receptors and neuronal activity by cannabinoids. At the synaptic level, recent studies have identified nicotinic receptor-dependent alterations in development of glutamatergic synapses (Lozada et al., 2012a; Morley and Mervis, 2013). Activation of postsynaptic α7-nicotinic receptors in cultured neurons or slice preparations promotes glutamatergic synapse formation, whereas knockout of the α7 subunit in mice decreases the number of dendritic spines (Lozada et al., 2012b) and glutamatergic synapses, suggesting that cholinergic signaling through this nicotinic receptor subtype is normally important in modulating the number of excitatory synapses. Inhibitory effects of CBs would be expected to affect formation of glutamatergic synaptic transmission and plasticity of neuronal networks. Further investigations focusing on the cannabinoid modulation of nicotinic receptor-

mediated developmental and synaptic changes will also help in understanding the mechanisms of cannabis-related synaptic plasticity during neuronal development. Another important subject is the selectivity of CB actions among various nicotinic receptor subtypes. In addition to α7-nicotinic receptor, the α4β2 is the predominant nicotinic receptor subtype in the CNS, and has been implicated in mediating both the positive-reinforcing and cognitive effects of nicotine (Tapper et al., 2004). A recent study in mutant mice revealed that the presence of the α4β2-nicotinic receptor subunit is both necessary and sufficient for the development of nicotine-induced tolerance and sensitization in vivo (Tapper et al., 2004). Using the whole-cell patch-clamp technique, Spivak et al., investigated the effects of AEA on human α4β2 nicotinic receptors expressed in SH-EP1 cells (Spivak et al., 2007). AEA, in the concentration range of 200 nM to 2 μM, reduced the maximal amplitudes and increased the desensitization of acetylcholine-induced currents. The effects of AEA could be neither replicated by the THC (1 μM) nor reversed by the SR-141716A (1 μM). The actions of AEA were apparent when applied extracellularly but not during intracellular dialysis. Kinetic analysis of AEA actions on α4β2 nicotinic receptor-mediated currents indicated that the first forward rate constant leading to desensitization increased nearly 30-fold as a linear function of AEA concentration. In contrast, the other three rate constants were unaltered by AEA suggesting that AEA raised the energy levels of the activated state (Spivak et al., 2007). Another study on α4β2 nicotinic receptors was conducted in rat thalamic synaptosomes where the acetylcholine-induced 86Rb þ effluxes have been reported to be carried mainly through α4β2 nicotinic receptors (Butt et al., 2008). Thus, Butt et al. (2008) tested the effects of AEA on the function of α4β2 nicotinic receptors in native brain membranes such as thalamic synaptosomes. In these preparation, AEA reversibly inhibited 86Rb þ efflux induced by 300 μM acetylcholine with an IC50 value of 0.972 μM. Pre-treatment with the CB1 receptor antagonist SR141716A (1 μM), the CB2 receptor antagonist SR144528 (1 μM), or pertussis toxin (0.2 mg/mL) did not alter the inhibitory effects of AEA. Inhibition of 86Rb þ efflux by AEA was not reversed by increasing acetylcholine concentrations and the specific binding of [3H]-nicotine was not altered by 30 μM AEA. To date, α7 and α4β2 receptor subtypes are affected by AEA and probably by other class of CBs as well. On the other hand, the function of muscle type nicotinic receptor has not altered CBs (Oz, 2006). Currently, there is not much evidence on the selectivity of CBs among different nicotinic receptor subunits. Undoubtedly, more detailed information on the subunit selectivity will be useful to elucidate mechanism of CB actions on nicotinic receptors.

3. Mechanisms of cannabinoid actions on nicotinic receptors Binding site of CBs on nicotinic receptors is currently unknown. However, in earlier studies using α7-nicotinic acetylcholine-5-HT3 chimeric receptors an attempt has been made to identify the binding site of AEA on α7-nicotinic receptor. The 5-HT3 receptor shares a high degree of homology with α7-nicotinic receptors. In the oocyte expression system, AEA inhibits 5-HT3 receptors with a potency that is at least one order of magnitude less than that of α7-nicotinic receptors (Oz et al., 2002, 2003). Thus in these studies, the IC50 values for AEA were 229 nM and 3.7 μM at α7-nicotinic and 5-HT3 receptors, respectively. The development of chimeric α7-nicotinic-5HT3 receptors that consist of N-terminal domain of the α7-nicotinic receptors and transmembrane domains and the carboxyl terminal of 5HT3 receptor (Eisele et al., 1993; Zhang et al., 1997) and the differential sensitivities of these ligand-gated ion channels to AEA provided an opportunity to evaluate the location of the AEA interaction with the α7-nAChR. When the effect of 300 nM

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AEA on α7-nicotinic-5-HT3 chimeric receptors was examined, it was found that AEA did not inhibit these currents significantly. The observed resistance of the chimeric receptor to AEA suggests that the binding site for AEA must be located either at or near one of the transmembrane domains or near the carboxyl terminal of this receptor. However, the interpretation of these results has been further complicated by recent studies indicating that the inhibition of 5-HT3 receptor by AEA and other phytocannabinoids also depends on the expression level of this receptor (Xiong et al., 2008;Yang et al., 2010a,b). Increasing the density of 5-HT3 receptors in Xenopus oocytes, significantly increased the potency (i.e. decreased the IC50 value) of CBs tested, suggesting that affinity for their binding sites increases at low expression levels. Increased receptor density has also been associated with enhancement of receptor desensitization. Therefore, conditions promoting desensitization of 5-HT3 receptors are also likely to potentiate the action of cannabinoids on these receptors (Xiong et al., 2008). At this time, it is not clear if these pharmacological properties of CBs can be generalized to other receptors such as nicotinic receptors. However, at least in case of cannabidiol, altering the density of α7-nicotinic receptor, did not affect cannabidiol inhibition of this receptors (Mahgoub et al., 2013). Nevertheless, further studies are needed to elucidate the mechanisms of the receptordensity and/or desensitization dependent actions of CBs. Apart from specific binding sites on the nicotinic receptors, CBs can accumulate and reach substantially high concentrations in biological membranes; thereby causing significant alterations in physico-chemical properties of membranes. In fact, the partition coefficient of AEA would be anticipated to be in the same order with that of AA acid to biological membranes (2–9  104, (Meves, 2008)). As a result, the membrane concentration of AEA would reach much higher levels than those estimated for intracellular or extracellular concentrations. In the case of 2-AG, another endocannabinoid, since most of the AA is located at the sn-2 position of the phospholipids, its levels exceed AEA levels in mammalian brain by 2–3 orders of magnitude (for a comprehensive review, see Sugiura et al. (2006)). Incorporation of these endocannabinoids into biological membranes can alter functional properties of integral membrane proteins such as nicotinic receptors. Results of earlier studies suggest that concentrations of CBs modulating the activity of nicotinic receptors appear to be within the range of 0.1 to 10 mM (depending on the cell type, expression system, subunit combinations, etc.). It is not known whether these concentrations can be achieved under physiological conditions. The concentrations of AEA in the rat brain have been reported to range from 2.5 to 29 pmol/g (Schmid et al., 2002). The concentration of AEA in rat and human plasma is in the nM range (0.7–8 nM and 4–20 nM, respectively (Giuffrida et al., 2000; Bojesen and Bojesen, 1994). 2-AG is found in gut and in brain at levels 170 times greater than that of AEA (Stella et al., 1997). However, as mentioned earlier, due to high partition coefficients, both endocannabinoids and other classes of CB receptor ligands can effectively accumulate in cell membranes and reach significantly higher concentrations. In this context, a recent study indicates that enhanced endocannabinoid levels not only alter the function of nicotinic receptors but also modulate the actions of alcohols and volatile anesthetics on α7-nicotinic receptors and probably other receptors (Oz et al., 2005; Jackson et al., 2008) suggesting that CBs, when accumulated in biological membranes, can affect the pharmacological actions of other hydrophilic compounds as well. It is noteworthy that cannabinoids, steroids, fatty acids, general anesthetics and alcohols, with varying degrees of lipophilic structures, also act as allosteric modulators (i.e., these molecules bind to sites topographically distinct from the ligand binding sites) of several structurally different ion channels including nicotinic receptors (Tillman and Cascio, 2003). Besides allosterism, another common feature of these lipophilic molecules is that they seem to

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affect preferentially the gating properties of both voltage and ligand-gated channels (for reviews, see Meves (2008) and Tillman and Cascio (2003)). It has been more than a century since Overton–Meyer rule stated that membrane permeability of any molecule depends on its hydrophobicity. Although the Overton–Meyer rule predicts the accessibility of hydrophobic molecules such as various lipids, fatty acids and endocannabinoids to their membrane-delimited actions and hence somewhat the potency of these molecules on their target proteins, it does not elucidate the action mechanism(s) of these molecules. A school of thought focuses on the effects of altering lateral pressure profiles on integral membrane proteins. It appears that these biophysical approaches help to describe how proteins sense the effects of hydrophobic molecules in membrane bilayers (Van den Brink-van der Laan et al., 2004). For example, hydrophobic mismatch between the lengths of the hydrophobic membrane-spanning domains and the bilayer thickness (Lundbaek, 2006), and changes in the lateral pressure profile of bilayer membranes (Cantor, 2001; Van den Brink-van der Laan et al., 2004) by partitioning of lipids or other hydrophobic molecules (Cantor, 2001) have been shown to modulate the functions of various ion channels and receptors. In line with these suggestions, in an earlier report, Bloom et al. showed that changes in the lipid order of synaptic membranes induced by cannabinoids might be a necessary property for their pharmacological activities (Bloom et al., 1997). Nevertheless, the effects of these lipophilic compounds on lateral membrane pressure and membrane thickness are not always observed, and other membrane properties are also likely to play roles in lipid–protein interactions (Lee et al., 2005). Another school of thought on the question of how lipophilic molecules such as CBs affect the function of ion channels focuses on the protein–lipid interface. It has been suggested that lipids such as fatty acids displace (Antollini and Barrantes, 2002) or interact (Garcia, 2004) with lipids and/or hydrophobic amino acids located at the specific lipid–protein interfaces of the ion channels (Barrantes, 2004), rather than inducing changes in bulk physico-chemical properties of membranes. Thus, although lipophilicity is an important feature determining the bioavailibilty of the drug at its action site, highly lipophilic molecules such as CBs may not need to change general biophysical characteristics of membranes to modulate the function of ion channels (Barrantes, 2004; Garcia, 2004). Extensive volume of work in the literature indicates that the bilayer is not simply an inert thin layer of lipid whose main function is to provide a barrier to ions (McIntosh and Simon, 2006). Following their insertion into fluid membrane bilayer, ion channels assume an energetically minimal conformational state leading to a stable structure. Importantly, the binding of ligand causes conformational changes on the structure of the proteins. It is thought that these conformation changes are associated with the alterations in the gating domains of the ion channels (Lee and MacKinnon, 2004; Lee et al., 2005; Garcia, 2004). The energetic requirements of these conformational changes depend significantly on the lipid environment in which they are immersed (Spivak et al., 2007). It is likely that, due to their high lipophilicity, cannabinoid receptor ligands can alter the physico-chemical characteristics of the lipid environment or bind to hydrophobic sites on the nicotinic receptors and regulate the functional properties of these receptors. Although the exact mechanisms of nicotinic receptor regulation by CBs are currently unknown, such interaction with nicotinic receptors is likely to mediate some of the pharmacological actions of CBs in the nervous system. References Acquas, E., Pisanu, A., Marrocu, P., Di, C.G., 2000. Cannabinoid CB(1) receptor agonists increase rat cortical and hippocampal acetylcholine release in vivo. Eur. J. Pharmacol. 401, 179–185.

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