These data thus suggest that AEA augments ventricular myocardial IKATP through a CB2 receptordependent pathway, which may underlie the antiarrhythmic and cardioprotective action of AEA; by contrast, AEA exerts no effect on another inward rectifier current IK1 in ventricular myocytes. Nevertheless, whether AEA causes an inhibitory effect on myocardial KATP channels in excised membrane patches remains to be determined. Endocannabinoid production can be induced when the cardiovascular system is functioning under deleterious conditions such as circulatory shock or hypertension; endocannabinoids are also involved in preconditioning by nitric oxide. Activation by endogenously released AEA under pathophysiological conditions may contribute to the cardioprotection afforded by sarcolemmal KATP channels. The difference in KATP channel responses between different cell types to endocannabinoids may be partly attributed to the distinct, tissue-specific molecular compositions of KATP channels or the cellular background in which the channels are expressed.Across the studies discussed above, the time course for endocannabinoid lipids and analogues to induce the potassium channel-modulating effects is generally slow, with a maximal response achieved after several minutes of continuous drug exposure. Moreover, there is no significant washout of the endocannabinoid effect upon perfusion with drug-free solution, unless the wash solution contains lipid-free BSA . In almost all of these studies, the experimentation was performed at room temperature, except the study by Gantz and Bean, where the experimentation was conducted at 37°C instead. Cannabinoids are lipid-soluble compounds, and dimethyl sulfoxide or 100% ethanol was chosen as a solvent to prepare aliquots of endocannabinoids at millimolar concentrations in these studies,cannabis grow systems with the final concentration of solvent during experiments consistently ≤ 0.1–- 0.15%. Gantz and Bean showed that the maximal inhibitory effect of 2-AG on the fast inactivating A-type K+ current IA could be measured within 1 min of drug exposure .

The more prompt response to endocannabinoids observed by Gantz and Bean may be attributed, in part, to the higher temperature at which their study was carried out.AEA can modulate the functions of ion channels other than potassium channels, such as TRP vanilloid type 1 channels, 5-HT3 receptors, nicotinic acetylcholine receptors, glycine receptors, and CaV and voltage-gated Na+ channels, in a manner independent of known cannabinoid receptors. Several studies are reviewed below to exemplify that, besides potassium channels, multiple ion channel types belonging to other ion channel families can also serve as molecular targets of endocannabinoids, which collectively manifests the relevance of direct modulation of various ion channels in mediating the biological functions of endocannabinoids.The TRP channel superfamily of non-selective, ligand-gated cation channels is involved in numerous physiological functions such as thermo- and osmosensation, smell, taste, vision, hearing pressure or pain perception. The endocannabinoid AEA is structurally related to capsaicin , the agonist for TRPV1 channels. It has been demonstrated by Zygmunt et al. that AEA induces vasodilation in isolated arteries in a capsaicin-sensitive manner and that the AEA effect is accompanied by release of calcitoningene-related peptide , a vasodilator peptide. This vasodilatory action of AEA is abolished by a CGRP receptor antagonist but not by the CB1 receptor antagonist SR141716A; moreover, CB1 and CB2 receptor agonists do not reproduce vasodilation caused by AEA. Additionally, AEA concentration-dependently elicits capsazepine -sensitive currents acquired from cells over expressing cloned TRPV1 channels in both whole-cell and excised patch modes. These findings thus suggest that AEA induces peripheral vasodilation by activating TRPV1 channels on perivascular sensory nerves independently of CB1 receptors and consequently causing the release of CGRP. AEA and other structurally related lipids may act as endogenous TRPV channel agonists or modulators to regulate various functions of primary sensory neurons such as nociception, vasodilation, and neurogenic inflammation.Low voltage-activated or T-type calcium channels, encoded by the CaV3 gene family, regulate the excitability of many cells, including neurons involved in nociceptive processing, sleep regulation and the pathogenesis of epilepsy; they also contribute to pacemaker activities.

The whole-cell currents of both cloned and native T-type calcium channels are blocked by sub-micromolar concentrations of AEA; this effect is prevented by inhibition of AEA membrane transport with AM404, suggesting that AEA acts intracellularly. AEA concentration-dependently accelerates inactivation kinetics of T-type calcium currents, which accounts for the reduction in channel activity. The inhibitory action of AEA on these CaV channels is independent of CB1/CB2 receptors and G proteins, and the inhibition is preserved in the excised inside-out patch configuration, implying a direct effect; furthermore, AEA has little effect on membrane capacitance, reflecting that its effects are unlikely attributed to simple membrane-disrupting mechanisms. Accordingly, it is postulated that AEA may directly target and modulate T-type calcium channels to elicit some of its pharmacological and physiological effects. High voltage-activated, dihydropyridine sensitive L-type calcium channels are involved in excitation-contraction coupling in skeletal, smooth, and cardiac myocytes as well as the release of neurotransmitters and hormones from neurons and endocrine cells. It has been demonstrated via biochemical assays that AEA is able to displace specific binding of L-type calcium channel antagonists to rabbit skeletal muscle membranes in a concentration-dependent manner, with the IC50 around 4–30 μM, supporting a direct interaction between AEA and L-type calcium channels. Furthermore, AEA suppresses the whole-cell currents of both native NaV and L-type calcium channels in rat ventricular myocytes in a voltage- and pertussis toxin-independent manner, indicating that the inhibitory effect of AEA does not require activation of Gi/o protein-coupled receptors like CB1 and CB2 receptors. Direct inhibition of NaV and L-type CaV channel function may account for some of the negative inotropic and antiarrhythmic effects of AEA in ventricular myocytes.GlyRs belong to the Cys-loop, ligand-gated ion channel superfamily that comprises both cationic receptors such as nAChRs and 5-HT3Rs and anionic receptors such as γ-aminobutyric acid type A receptors and GlyRs. GlyRs are distributed in brain regions involved in pain transmission and reward, and they are thought to play a role in the analgesia process and drug addiction. AEA, at pharmacologically relevant concentrations , directly potentiates the function of recombinant GlyRs expressed in oocytes and native GlyRs present in acutely isolated rat ventral tegmental area neurons through an allosteric, CB1 receptor-independent mechanism.

The stimulatory effect of AEA on GlyRs is selective, as neither the GABA-activated current in VTA neurons nor the recombinant α2β3γ2 GABAAR current in oocytes is affected by AEA treatment.The homomeric α7 receptor is one of the most abundant nAChRs in the nervous system and it is involved in pain transmission, neurodegenerative diseases, and drug abuse. The endocannabinoid AEA has been shown to inhibit nicotine-induced currents in Xenopus oocytes expressing cloned α7 nAChRs; the inhibition is concentration-dependent with an IC50 of 229.7 nM and noncompetitive. In addition, pharmacological approaches using specific inhibitors uncovered that the inhibitory effect of AEA on α7 nAChRs does not require CB receptor activation, G protein signaling, AEA metabolism, or AEA membrane transport, suggesting that AEA inhibits the function of neuronal α7 nAChRs expressed in Xenopus oocytes via direct interactions with the channel. AEA is structurally similar to other fatty acids such as arachidonic acid and prostaglandins; it is possible that AEA and other fatty acids that are capable of modulating nAChRs share some common mechanisms of action to control the channel function.It is well established that potassium channels are important players in controlling the duration, frequency, and shape of action potentials, thereby controlling cell excitability. As described above, the endocannabinoid AEA is capable of exerting CB1/CB2 receptor-independent functional modulation of a variety of potassium channels, including native BK , Ito, delayed rectifier, KATP and TASK-1 channels, as well as cloned TASK-1, Kv4.3, Kv3.1, Kv1.2 ,and Kv1.5 channels . Moreover, native neuronal IA, pancreatic β–cell delayed rectifier and KATP, and atrial myocardial delayed rectifier potassium channels are subject to modulation by another endocannabinoid 2-AG, also in a CB receptor-independent manner . Likewise, for TRPV1 channels, ligand-gated ion channels such as cloned and native GlyRs, cloned α7 nAChRs and native 5-HT3Rs , plus voltage-gated ion channels such as native NaV , native L-type CaV, and native and cloned T-type CaV channels,ebb and flow tables the functional modulation elicited by AEA does not require activation of CB receptors . Interestingly, in the majority of studies reviewed in this article, the CB receptor-independent modulatory effects of AEA are induced only when endocannabinoids are introduced extracellularly to the ion channel targets that include cloned Kv1.2, hKv1.5, Kv3.1, and hKv4.3 channels heterologously expressed, and native delayed rectifier Kv channels in aortic vascular smooth muscle cells and cortical astrocytes, whereas in several reports endocannabinoids only alter ion channel function when administrated at the cytoplasmic side of the membrane. These observations imply the presence of distinct interaction sites or mechanisms of action, which may be attributable to differences in the types of ion channels or endocannabinoids investigated, cell models/cellular environments channels being exposed to, or experimental protocols adopted. On the other hand, although membrane environment seems to be critical for the regulation of signal transduction pathways triggered by G protein-coupled receptors like CB1 receptors, current evidence does not support an involvement of changing membrane fluidity or altering lipid bilayer properties in mediating the CB receptor-independent actions of AEA on ion channels.

Besides, it is worth noting that, unlike 2-AG, which is entirely localized in lipid rafts in dorsal root ganglion cells, most of AEA is found in non-lipid raft fractions of the membrane. It is therefore less likely that changes in membrane fluidity serve as a primary mechanism of action responsible for AEA’s CB receptor-independent effects. Lipid signals like endocannabinoids and structurally related fatty acids may modify gating of voltage-gated ion channels through a direct action on the channel via a membrane lipid interaction. A model for direct interactions between ion channel proteins and endocannabinoids is further supported by identification of specific residues in several channel proteins crucial for the CB receptor-independent modulatory actions exerted by endocannabinoids. For example, AEA may directly interact with, and in turn be stabilized by, a ring of hydrophobic residues formed by valine 505 and isoleucine 508 in the S6 domain around the ion conduction path of the hKv1.5 channel, thereby plugging the intracellular channel vestibule as a high potency open-channel blocker and suppressing the channel function. Molecular dynamic simulations have also helped reveal novel interactions between AEA and the TRPV1 channel on a molecular level, suggesting that AEA enters and interacts with TRPV1 in a location between the S1-S4 domains of the channel via the lipid bilayer.Brief interventions have empirical support for acutely reducing alcohol use among non-treatment seeking heavy drinkers. For example, randomized clinical trials of brief interventions have found favorable results among heavy drinkers reached through primary care , trauma centers and emergency departments . Brief interventions also have shown effectiveness in reducing alcohol use in non-medical settings among a young adult college population . Given this sizable evidence base, there is considerable interest in understanding the underlying mechanisms toward optimizing this approach. Neuroimaging techniques allow for the examination of the neurobiological effects underlying behavioral interventions, probing brain systems putatively involved in clinical response to treatment. To date, one study has examined the effect of a motivational interviewing-based intervention on the neural substrates of alcohol reward . In this study, neural response to alcohol cues was evaluated while individuals were exposed to change talk and counter change talk , which are thought to underlie motivation changes during psychosocial intervention. The authors report activation in reward processing areas following counter change talk, which was not present following exposure to change talk . Feldstein Ewing and colleagues have also probed the nature of the origin of change talk in order to better understand the neural underpinnings of change language . In this study, binge drinkers were presented with self-generated and experimenter-selected change and sustain talk. Self-generated change talk and sustain talk resulted in greater activation in regions associated with introspection, including the interior frontal gyrus and insula, compared to experimenter elicited client language . These studies employed an active ingredient of MI within the structure of the fMRI task, thus allowing for a more proximal test of treatment effects. Neuroimaging has also been used to explore the effect of psychological interventions on changes in brain activation that are specifically focused on alcohol motivation. For example, cue-exposure extinction training, a treatment designed to prevent return to use by decreasing conditioned responses to alcohol cue stimuli through repeated exposure to cues without paired reward, has also been evaluated using neuroimaging .