Effects of cannabinoids on ligand-gated ion channels

Effects of cannabinoids on nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are cationic channels belonging to the Cys-loop superfamily of ligand-gated ion channels and their opening is controlled by the endogenous neurotransmitter ACh or exogenous ligands such as nicotine (Albuquerque et al., 2009; Dineley et al., 2015). They are homo or hetero-pentameric complexes comprised of α1-10, β1-6, δ, and γ subunit combinations and mediate fast cholinergic neurotransmission in both central and peripheral nervous systems and are expressed in non-neuronal cells as well (Albuquerque et al., 2009; Zoli et al., 2015). The homomeric α7-nAChR subtype plays an important role in synaptic plasticity and various disease pathologies including pain, neuroinflammation, and neurodegenerative diseases such as Alzheimer and Parkinson diseases (Oz et al., 2013; Lorke et al., 2016; Oz et al., 2016; Borroni and Barrantes, 2021; Mizrachi et al., 2021; Papke and Horenstein, 2021).

In vivo studies, WIN55,212-2, non-selective cannabinoid receptor agonist, has been shown to impair memory-related effects of nicotine in male rats (Robinson et al., 2010) and mice (Biala and Kruk, 2008). In Xenopus oocytes expressing α7-nACh receptors, AEA and 2-AG noncompetitively inhibited α7-nACh receptor-mediated currents with IC₅₀ values of 229 and 168 nM, respectively (Oz et al., 2003; Oz et al., 2004b). In line with these findings, radioligand binding studies indicated that AEA, at concentration range of 30-300 μM, does not alter specific binding of [³H]nicotine in human frontal cortex (Lagalwar et al., 1999) and rat thalamic membranes (Butt et al., 2008), further suggesting a noncompetitive nature of AEA effect on nACh receptors. Inhibition of α7-nACh receptor by AEA was not reversed by either SR 141716A (Rimonabant), specific CB1 antagonist, or SR 144528, CB2 antagonist and was not sensitive to pertussis toxin treatment (Oz et al., 2003). In line with earlier findings, arachidonic acid (AA), a fatty acid moiety of AEA (Vijayaraghavan et al., 1995), but not ethanolamine or glycerol, inhibited the function of α7-nACh receptors suggesting that it was the intact endocannabinoid and not the metabolite AA that altered the function of α7-nACh receptor. In an in vivo study, Baranowska et al., demonstrated that, in the presence of AM2521, a CB1 antagonist, methanandamide, a metabolically stable analog of AEA inhibited nicotine-induced tachycardiac responses mediated by the activation of α7-nACh receptors on the cardiac postganglionic sympathetic neurons of urethane anesthetized rats (Baranowska et al., 2008). In another in vivo study, increased endocannabinoid levels modulate the somatic signs of nicotine withdrawal by a mechanism that does not involve CB1 receptors (Merritt et al., 2008).

Effects of synthetic cannabinoids on the functions of nACh receptors have been tested in a few studies. In Xenopus oocytes, WIN55,212-2 was ineffective up to concentrations of 10 μM, but CP55940 inhibited α7-nACh receptor with an IC₅₀ value of 2.7 µM (Oz et al., 2004b). On the other hand, in cultured rat trigeminal ganglion neurons, patch-clamp studies indicated that native nicotinic receptor was inhibited by WIN55,212-2 with an IC₅₀ value about 3 µM in a cannabinoid receptor independent manner (Lu et al., 2011).

Among the phytocannabinoids tested, THC and cannabinol up to 10 µM were ineffective (10-20% inhibition) on α7-nACh receptors expressed in Xenopus oocytes (Oz et al., 2004b; Mahgoub et al., 2013). Similarly, THC (1 µM) did not affect the amplitudes of currents through α4β2-nACh receptors (Spivak et al., 2007). CBD, on the other hand, at relatively high concentration modulates the function of nACh receptors. In earlier studies, CBD was reported to decrease the amplitudes of miniature end-plate potentials in frog neuromuscular junction suggesting an effect on the postsynaptic nACh receptors (Turkanis and Karler, 1986). Later studies indicated that CBD inhibits human α7-nACh receptors with an IC₅₀ of 11.3 µM (Mahgoub et al., 2013; Oz et al., 2014) and suppresses choline-induced inward currents in rat hippocampal interneurons. CBD has also been shown to inhibit nicotine-induced [³H]-norepinephrine release in rat hippocampal slices (Mahgoub et al., 2013), and reduce withdrawal symptoms in nicotine-dependent rats (Smith et al., 2021) further suggesting an interaction between CBD and nicotinic receptors. Notably, Inhibitory effect of CBD was time-dependent with 50% inhibition occurring in 2.1 min sustained CBD application, voltage-independent, and non-competitive, i.e., while the EC₅₀ of ACh remained unaltered, maximal ACh responses were significantly decreased (Mahgoub et al., 2013). In line with these findings, specific binding of [¹²⁵I] α-bungarotoxin binding was not affected by CBD.

In addition to α7-nicotinic receptor, the α4β2 is the major nACh receptor subtype in the CNS and has been implicated in mediating both the positive-reinforcing and cognitive effects of nicotine (Tapper et al., 2004). The presence of the α4β2-nACh receptor subunit appears to be 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., showed that AEA, in the concentration range of 200 nM to 2 μM, reduced the maximal amplitudes and increased the desensitization of acetylcholine-induced currents mediated by human α4β2 nACh receptors expressed in SH-EP1 cells (Spivak et al., 2007). The effects of AEA could be neither reversed by the SR141716A (1 μM) nor replicated by the THC (1 μM). Interestingly, AEA exerted inhibitory effect when applied extracellularly but not during intracellular dialysis. Kinetic analysis of the effect of AEA on α4β2 nACh receptor-mediated currents demonstrated that the first forward rate constant leading to desensitization increased nearly 30-fold as a linear function of AEA concentration and the energy levels of the activated state were raised by AEA (Spivak et al., 2007). Another study on α4β2 nACh receptors utilized rat thalamic synaptosomes where the ACh-induced ⁸⁶Rb⁺ effluxes carried mainly through native α4β2 nACh receptors were reversibly inhibited by AEA with an IC₅₀ of 0.9 µM in a noncompetitive manner (Butt et al., 2008). Inhibitory effect of AEA was not altered by pretreatments with the CB1 receptor antagonist SR141716A (1 μM), the CB2 receptor antagonist SR144528 (1 μM), and PTX. Although α7 and α4β2 receptor subtypes are affected by AEA, the function of muscle type nicotinic receptor is not altered by AEA (Oz, 2006). The results of key studies investigating the effects of cannabinoids on nACh receptors are summarized in Table 1.

Effects of cannabinoids on serotonin type 3 (5-HT₃) receptors.

There are seven classes of 5-HT receptors (5-HT1–5-HT7). 5-HT₃ receptor is the only ionotropic cation-selective ion channel which belongs to the superfamily of Cys-loop ligand-gated ion channels (Barnes et al., 2009; Lummis, 2012), remaining 5-HT receptor classes are G-protein-coupled receptors. To date, there are five 5-HT₃ receptor genes termed A-E. Only 5-HT_3A subunit can assemble as a functional homo-pentameric channel and 5-HT_3A is an obligate participant in all other 5-HT₃ receptor complexes (Lummis, 2012). Activation of 5-HT₃ receptors depolarizes neurons and mediates fast, excitatory synaptic transmission in the central and peripheral nervous systems. The highest number of 5-HT₃ receptor binding sites occurs in the area postrema and solitary tract nucleus (Morales and Wang, 2002). In addition, large numbers of 5-HT₃ receptors are found in the gastrointestinal system where activation of 5-HT₃ receptors are shown to alter gastrointestinal motility and to regulate the vomiting reflex (Sanger and Andrews, 2006), and they also appear to play a role in irritable bowel syndrome, visceral pain and inflammation (Faerber et al., 2007; Thompson and Lummis, 2007; Machu, 2011).

Effects of cannabinoids on the functions of 5-HT₃ receptors were first demonstrated in nodose ganglion neurons (NGNs) and Xenopus oocytes (Fan, 1995; Oz et al., 1995). In NGNs, AEA, WIN55,212-12, CP55,940 and its nonpsychoactive enantiomer CP56,667 inhibited 5-HT₃ receptors with IC₅₀ values of 94 nM, 190 nM, 310 nM and 1.6 μM, respectively (Fan, 1995). Subsequent studies on 5-HT₃ receptors expressed in Xenopus oocytes showed that AEA directly inhibits the function of 5-HT₃ receptors with an IC₅₀ value of 3.7 μM (Oz et al., 2002a). The inhibition developed gradually, reaching steady-state within 10–20 min, and it was non-competitive with respect to 5-HT. In both NGNs and Xenopus oocytes, agents known to modulate G-protein functions such as PTX treatments and intracellular applications of GDP-β-S, the nonhydrolyzable analogue of GTP, as well as applications of agents known to modulate intracellular cAMP levels did not alter the extent of AEA inhibition of 5-HT₃ receptors (Fan, 1995; Oz et al., 2002a). In another study, using patch clamp technique in excised outside-out patch mode, it was demonstrated that cannabinoid receptor agonists THC, WIN55,212-2, AEA, JWH-015, LY320135, and CP55,940 inhibited the function of 5-HT₃ receptors expressed in HEK-293 cells with IC₅₀ values of 38, 104, 130, 147, 523, and 648 nM, respectively (Barann et al., 2002). Similarly, inhibition of 5-HT₃ receptor mediated currents in rat trigeminal ganglion neurons by WIN55,212-2 was time and concentration-dependent (IC₅₀ = 0.1 µM), and not reversed by cannabinoid receptor antagonists (Shi et al., 2012). The inhibition of 5-HT₃ receptors by WIN55,212-2 in HEK-293 cells (Barann et al., 2002) and AEA in Xenopus oocytes (Oz et al., 2002a) was not altered by SR141716A, and their effects were noncompetitive. The cannabinoid receptor ligands [³H]-SR141716A and [³H]-CP55,940 did not specifically bind to parental HEK-293 cells. In competition experiments on membranes of HEK-293 cells transfected with the 5-HT₃ receptor cDNA, WIN55,212-2, CP55,940, AEA and SR141716A did not affect the binding of [³H]-GR65630, a specific ligand for 5-HT3 receptors (Barann et al., 2002).

Interestingly, potencies of AEA actions on 5-HT₃ receptors differ significantly between cell lines such as HEK-293 (Barann et al., 2002) and Xenopus oocytes (Oz et al., 2002a). The mechanisms underlying these seemingly discrepant results were studied in a recent study (Xiong et al., 2008). It was found that differences in the potencies of AEA inhibition of 5-HT₃ receptors between Xenopus laevis oocytes and HEK-293 cells were mainly due to different levels of steady-state receptor density at the cell surface. The magnitude of AEA inhibition was inversely correlated with the expression level of receptor protein; i.e., increasing surface receptor expression decreased the magnitude of AEA inhibition. In line with these findings, pretreatment with actinomycin D, an inhibitor of transcription process, decreased the amplitude of current activated by maximal 5-HT concentrations and increased the magnitude of AEA inhibition. AEA accelerated 5-HT₃ receptor desensitization time in a concentration-dependent manner without significantly changing receptor activation and deactivation time. The desensitization time was correlated with the AEA-induced inhibition and mean 5-HT current density. Applications of 5-hydroxyindole and nocodazole, a microtubule disruptor, significantly slowed 5-HT₃ receptor desensitization and reduced the magnitude of AEA inhibition. Collectively, these findings suggested that 5-HT₃ receptor density at the steady state regulates receptor desensitization kinetics and the potency of AEA-induced inhibiting effect on the receptors. Thus, it appears that the differences in IC₅₀ values for AEA inhibition of 5-HT₃ receptor in Xenopus oocytes (3.7 μM; (Oz et al., 2002a); and HEK-293 cells (130 nM; (Barann et al., 2002)) are mainly due to the different receptor expression levels among various cell types.

In vivo studies, WIN55,212-2 and CP55,940 inhibited the responses mediated by 5-HT₃ receptors located on the terminals of cardiopulmonary afferent C-fibers in anesthetized and SR141716A pretreated rats (Godlewski et al., 2003). Similarly, CBD inhibited the reflex bradycardia induced by the 5-HT₃ receptor agonist phenylbiguanide in spontaneously hypertensive rats (Kossakowski et al., 2019). In another in vivo study, the inhibitory effect of WIN55,212-2 on cocaine-induced locomotor hyperactivity was reported to be mediated by the inhibition of 5-HT₃ receptors (Przegaliński et al., 2005). In a recent study, mice deficient in CB1 and CB2 receptors were treated with THC and AEA, in the presence of the 5-HT₃ antagonist ondansetron (Rácz et al., 2008). AEA induced analgesia, but not catalepsy, is completely blocked by ondansetron, suggesting that 5-HT₃ receptors are involved in cannabinoid-induced analgesia in a manner independent of known cannabinoid receptors (Rácz et al., 2008).

Both THC and CBD were shown to directly interact with 5-HT₃ receptors expressed in HEK-293 cells (Barann et al., 2002; Xiong et al., 2011b), Xenopus oocytes, and NGNs (Yang et al., 2010a; Yang et al., 2010b; Xiong et al., 2011b). Depending on the expression levels of the receptor, THC and CBD inhibited human 5-HT_3A receptors with respective IC₅₀ values of 1.2 µM and 0.6 µM in Xenopus oocytes (Yang et al., 2010a; Yang et al., 2010b) and 119 nM and 329 nM in HEK-293 cells (Xiong et al., 2011b). Inhibition by these cannabinoids was voltage-independent, non-competitive, and increased with sustained application (preincubation) time. In line with non-competitive nature of their effect, THC and CBD did not alter the specific [³H]GR65630 binding on 5-HT_3A receptors (Yang et al., 2010a; Yang et al., 2010b). Importantly, slowing the receptor desensitization by pharmacological agents such as 5-hydroxyindole and nocodazole or mutations such as R427L markedly decreased the extent of 5-HT₃ receptor inhibition by CBD (Xiong et al., 2011b). Similarly, increasing the expression level and density of 5-HT₃ receptors on the cell membrane caused a noticeable decrease of receptor desensitization and the potency of THC on 5-HT₃ receptor (Yang et al., 2010b). Finally, 5-HT3 and CB1 receptors colocalize in hippocampal and dentate gyrus interneurons (Morales and Bäckman, 2002) and in other brain regions as well (Morales et al., 2004). Therefore, it is likely that the actions of cannabinoids including AEA and THC within the same cell can be mediated by simultaneous dual actions of cannabinoids on cannabinoid and 5-HT₃ receptors. The results of key studies investigating the effects of cannabinoids on 5-HT₃ receptors are summarized in Table 2.

Effects of cannabinoids on ionotropic glutamate receptors

Glutamate mediates most excitatory neurotransmission in the mammalian central nervous system through activation of metabotropic, G protein–coupled glutamate receptors, and ionotropic glutamate receptors, which are cation-selective ligand-gated ion channels. Ionotropic glutamate receptors are divided into different functional classes, namely α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, kainate receptors, N-methyl-D-aspartate (NMDA) receptors, and GluD (Δ) receptors (Hansen et al., 2021). In vivo studies, synthetic cannabinoid HU211 protected against tremorogenic, convulsive, and lethal effects of NMDA in mice and acted as a functional antagonist in radioligand binding studies (Feigenbaum et al., 1989). Similarly, NMDA-induced toxicity was reversed by THC, but not WIN55,212-2 in SR141716A insensitive manner in AF5 cells (Chen et al., 2005). AEA (1-10 µM), but not THC, potentiated NR1/NR2A NMDA receptor-mediated currents in Xenopus oocytes and increased NMDA-induced intracellular Ca²⁺ transients in the presence of SR141716A in rat cortical, cerebellar, and hippocampal slices (Hampson et al., 1998). 2-AG (5 µM) enhanced NMDA-evoked currents in a CB1 and TRPV1 receptor-independent manner through activation of PKC/Src signaling pathway (Yang et al., 2014). In studies of blood pressure control in rats, it was found that AEA produces a pressor effect in the presence of SR141716A and this increase in pressure was partially reduced by the NMDA receptor antagonist MK-801, suggesting a possible direct interaction between NMDA receptors and AEA with relevance to central control of blood pressure (Malinowska et al., 2010).

CBD inhibited AMPA-glutamate receptor-mediated evoked excitatory postsynaptic currents and suppressed the frequency and amplitudes of miniature excitatory postsynaptic currents in mice hippocampal neurons (Yu et al., 2020). Furthermore, in HEK-293 cells, CBD was shown to inhibit recombinant GluA1 currents with IC₅₀ of 22.5 µM and facilitate the deactivations of GluA1 and GluA2 receptors and slow the recovery of GluA1 receptor from desensitization (Yu et al., 2020). In line with these findings, CBD alleviate behavioral hyperactivity and hippocampal c-Fos expression in Gria1−/− mice which is a model sensitive to drugs that downregulate glutamatergic transmission such AMPA antagonists (Aitta-Aho et al., 2019). AEA, at high concentrations, inhibits homomeric GluR1 and GluR3 subunit-mediated currents with IC₅₀ of 161 µM and 143 μM, respectively (Akinshola et al., 1999). AEA inhibited heteromeric GluR1/3 and GluR2/3 receptor subunits with similar IC₅₀ value of 148 µM and 241 μM, respectively.

Effects of cannabinoids on glycine receptors

Glycine receptors are inhibitory, anion-selective Cys-loop ligand-gated ion channels that mediate fast inhibitory neurotransmission in the spinal cord, brainstem and retina (Lynch, 2009). There are four known glycine receptor α subunits (α1–α4) and a single β subunit in vertebrates. Functional receptors assemble as homo-pentamers of α subunits, or as hetero-pentamers of α and β subunits (Burgos et al., 2016). Malfunctions of glycine receptors have been associated with a range of neurological disorders including hyperekplexia, temporal lobe epilepsy, autism, breathing disorders, and chronic inflammatory pain (Lynch et al., 2017).

In earlier studies, co-application of glycine with AEA or 2-AG (0.2-2 µM), independent of activation of CB1 and TRPV1 receptors, markedly inhibited peak amplitudes and accelerated onset and desensitization of high (100 µM) glycine-activated currents in pyramidal neurons isolated from the hippocampus of neonatal rats (Lozovaya et al., 2005). In contrast to endocannabinoids, WIN55,212-2 (1 µM) did not significantly affect peak amplitudes but significantly accelerated the desensitization as well as onset of currents activated by high concentrations (100 µM) of glycine (Lozovaya et al., 2005; Yatsenko and Lozovaya, 2007) but potentiated the amplitudes of currents at low (40 µM) glycine concentrations (Iatsenko et al., 2007). Other cannabinoids such as HU-210 (Yang et al., 2008; Demir et al., 2009; Xiong et al., 2012b) and ajulemic acid (Ahrens et al., 2009b) also act as positive allosteric modulators of α1 glycine receptors.

Co-application of AEA (1-10 µM) with low (EC₁₀; 3-10 µM) concentrations of glycine potentiated the glycine-induced currents mediated by homomeric α1, α2, and α3 glycine receptors expressed in HEK-293 cells (Yang et al., 2008; Yévenes and Zeilhofer, 2011), Xenopus oocytes, and CNS neurons (Hejazi et al., 2006) in a CB receptor-independent manner. AEA-induced potentiation of these three glycine receptor subtypes was reduced by mutating a conserved intracellular lysine residue (K385A), whereas potentiation of α1 receptor induced by N-arachidonoyl-glycine was reduced by mutating loop 2 (A52), transmembrane 2 (G254) or intracellular (K385) amino acids (Yévenes and Zeilhofer, 2011). In another study, AEA (1-30 µM) substantially increased the amplitude of glycine-activated currents in cultured rat spinal neurons (Xiong et al., 2012b). The effect reached a steady-state within 5 min continuous AEA application and markedly diminished by increasing glycine concentrations. In HEK-293 cells, AEA potentiation of homomeric α1 and α3 receptors was significantly higher than α2 glycine receptors. While S296A and S307A mutations in transmembrane three domains of α1 and α3 receptors markedly decreased the potentiation by sustained (continuous), but not simultaneous co-application of AEA, A303S mutation increased the potentiation of α2 glycine receptors by AEA. The S296A mutation also decreased the extent of α1 receptor potentiation by other cannabinoids such as CBD, THC, and HU-210 (Xiong et al., 2012b). Importantly, the authors of this study also showed in control studies that these mutations have no significant effect on the pharmacological properties of glycine receptors. Furthermore, removal of hydroxy or oxygen groups in AEA and THC significantly reduced the potentiating effects of these compounds on α1 glycine receptor suggesting that these groups are important for their effects on glycine receptor. In outside-out patches in CHO cells expressing human α1 glycine receptor, continuous application of 2-AG (1 µM) inhibited the peak amplitudes, decreased the rise time, accelerated the desensitization rate, and slowed down the deactivation of the receptor activated by glycine at the level of EC₅₀ (40-70 µM). Notably, inhibitory effect of AEA was significantly less at low (20 µM) and high (1 mM) concentrations of glycine (Lozovaya et al., 2011). In further studies mimicking synaptic stimulation in outside-out patches from CHO cells by applying high concentration of glycine (1 mM) for 2 ms duration at 10 Hz frequency, application of 2-AG markedly reduced the amplitudes of glycine-induced currents. Similarly, in hypoglossal motoneurons of CB1 knock-out mice, 2-AG abolished the facilitation of synaptic glycinergic currents induced by repetitive (10-20 Hz) stimulations indicating that endocannabinoids can modulate synaptic transmission under physiological conditions in a cannabinoid-receptor-independent manner (Lozovaya et al., 2011).

CBD and THC have been shown to potentiate glycine receptor-mediated currents in a cannabinoid receptor-independent manner in Xenopus oocytes expressing homomeric α1 and heteromeric α1β glycine receptors, and in acutely isolated CNS neurons (Hejazi et al., 2006; Xiong et al., 2011a; Wells et al., 2015). CBD was reported to act as positive allosteric modulator of α1 and α1β1 glycine receptors expressed in HEK-293 cells with EC₅₀ values of 12.3 µM and 18.1 µM, respectively (Ahrens et al., 2009a). Notably, at higher concentration range, CBD directly acts as agonist of α1 and α1β1 glycine receptors with respective EC₅₀ values of 132.4 µM and 144.3 µM. The mutation of the α1 subunit transmembrane two serine 267) residue to isoleucine abolished both co-activation and direct activation of glycine receptor by CBD (Foadi et al., 2010). However, THC-induced potentiation was not affected when the S267Q mutant α1 glycine receptors were expressed in Xenopus oocytes (Hejazi et al., 2006). In subsequent studies, it was shown that continuous application, but not co-application, of THC (1 µM) for about 5 min caused a gradually developing marked potentiation (up to 1,200%) of glycine activated currents in HEK-293 cells expressing α1, α3, and α1β1, but not α2, subunits of glycine receptor (Xiong et al., 2011a). Notably, S296 residue of the α1 and the S307 of the α3 subunits have been shown to be critical for the potentiation of glycine receptors by continuous, but not simultaneous, THC application (Xiong et al., 2011a; Xiong et al., 2012b). Conversely, substitution of the corresponding residue, Ala303 of the α2 subunit with a serine converted the α2 subunit from a low to high THC sensitivity. Similar to THC, the CBD, dehydroxyl-CBD, and dehydroxyl-THC have also been shown to potentiate glycine-activated currents of α1 and α3 subunits (Xiong et al., 2011a; Xiong et al., 2012a; Xiong et al., 2012b). Further studies indicated that CBD and dehydroxyl-CBD suppress persistent inflammatory and neuropathic pain by potentiating α3 glycine receptor-mediated currents in HEK-293 cells and cultured spinal neurons and the CBD suppression of neuropathic pain was reversed in α3-knock-out mouse (Xiong et al., 2012a). In subsequent studies, dehydroxyl-CBD was reported to suppress inflammatory pain and potentiate α1-glycine receptors by interacting with S296 amino acid residue. Accordingly, analgesic effect of dehydroxyl-CBD was reversed in the transgenic mice carrying S296A mutation, which also blocks DH-CBD-induced potentiation of glycine-activated currents in neurons of the spinal cord dorsal horn in mutant mouse (Lu et al., 2018). In another study, dehydroxyl-CBD rescued functional deficiency of glycine receptor and exaggerated acoustic and tactile startle responses in mice bearing point mutations in α1 glycine receptors that are responsible for a hereditary startle-hyperekplexia disease (Xiong et al., 2014). Recently, cannabinoids such as THC and CBD were reported to rescue cocaine-induced seizures by restoring brain glycine receptor dysfunction in a cannabinoid-receptor independent manner (Zou et al., 2020b). The results of key studies investigating the effects of cannabinoids on glycine receptors are summarized in Table 3.