Ion channels are macromolecular pores in cell membranes that play a principal role in regulating the electrical properties of membranes and cellular excitability, thence enabling neurotransmission, sensory transduction, cognitive function, heart rhythm, motor movement, hormone secretion, reproduction, stress adaptation, and cell protection, processes crucial to sustain life. Ion channel proteins are subject to post translational modulation mediated by enzymes and cellular messengers . Notably, recent evidence has revealed that a variety of ion channel types are functionally modulated by cannabinoids, a structurally heterogeneous group of lipid-soluble compounds made naturally in plants or in animal cells which have emerged as prominent modulators of diverse physiological and pathological processes. Endocannabinoids refer to a class of signaling lipids consisting of amides, esters, and ethers of longchain polyunsaturated fatty acids that are synthesized naturally from lipid precursors in plasma membranes of animal organisms. Structurally distinct from the cannabinoids produced in the cannabis plant, endocannabinoids mimic the activity of Δ9 – tetrahydrocannabinol , the major psychoactive ingredient of cannabis. Endocannabinoids are part of the endocannabinoid system that consists of endocannabinoids, their biosynthesizing and biodegradative enzymes, the cannabinoid type 1 and type 2 receptors, and the cannabinoid uptake transporter.
The endocannabinoid system functions as a homeostatic regulator in essentially all organ systems for physiological processes including neurodevelopment,ebb and flow tray synaptic transmission, learning and memory, nociception, stress and emotions, immunomodulation, hormone secretion, food intake and energy balance, digestive tract motility and secretion, reproductive function, and bone mass, among others. In addition to the physiological roles, elements of the endocannabinoid system may serve as potential therapeutic targets in the pathological conditions. Indeed, pharmacological manipulation of the endocannabinoid system has been shown to induce antinociceptive, anticonvulsive, anxiolytic, and anti-inflammatory effects, mitigating the symptoms or progression associated with different diseases. N-Arachidonoylethanolamine , 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether represent the most notable endocannabinoids. AEA has widespread actions in the brain: it influences learning and memory in the hippocampus, modulates locomotor activity, food intake, and the reward pathway in the basal ganglia and hypothalamus, and renders antinociception in the spinal cord and supra spinal sites. Besides the central nervous system, AEA is also produced in the peripheral tissues where it exerts local effects such as regulation of vascular tone and control of embryo transport/implantation in the female reproductive tract. While many of AEA’s effects involve activation of CB1 or CB2 receptors, pharmacological evidence nonetheless suggests that additional targets for cannabinoids exist. For example, orphan G protein-coupled receptors , peroxisome proliferator-activated receptors , equilibrative nucleoside transporter-1 , serotonin receptor sub-types, transient receptor potential channels, and a variety of voltage-gated ion channels have all been suggested to mediate the effects of cannabinoids as their direct molecular targets.
The existence of additional targets of cannabinoids beyond CB1 and CB2 receptors reveals the complexity of the endocannabinoid system, manifesting an extensive endocannabinoid signaling network in homeostatic regulation and highlighting the promising prospect of targeting elements in this system for therapeutic interventions. In this review, I summarize evidence concerning the canonical CB receptor-independent actions of endocannabinoids on ion channels, with a primary focus on the interactions between potassium channels and AEA, one of the principal endocannabinoids. The aim is to put into perspective open questions that remain to be addressed for better understanding of the physiological mechanisms as well as potential medicinal uses of cannabinoids in human health and disease.The potassium channel super family exhibits a broad diversity that encompasses at least 70 channel members in mammals. Potassium channels are present in virtually all types of cells in all organisms, where they are involved in a multitude of physiological functions. These channels are important for setting the resting membrane potential, keeping fast action potentials short, terminating periods of intense activity, timing the interspike intervals during repetitive firing, and lowering the effectiveness of excitatory inputs on a cell. Potassium channels are involved in regulation of neurotransmission, heart rate, muscle contraction, hormone release, and cell survival, among others, and hence represent attractive drug targets for the development of new therapeutic strategies for cancer as well as metabolic, neurological, and cardiovascular disorders.
The endocannabinoid AEA belongs to a class of signaling lipids consisting of amides of long-chain polyunsaturated fatty acids. AEA is widely expressed in the human body and has been implicated in multiple physiological and pathophysiological processes, such as vascular tone, embryonic development, motor functions, pain reduction, cognition functions, sleep, immunomodulation, neuroprotection, cardioprotection, feeding and appetite, obesity, drug abuse, neurodegenerative diseases, and mood disorders. Many of AEA’s biological actions result from binding and activating the inhibitory Gi/o protein coupled CB1 and CB2 receptors. For instance, CB1 receptor-mediated presynaptic depression likely involves activation of G protein-gated inwardly rectifying potassium and A-type potassium channels through a CB1 receptor dependent signaling mechanism. However, AEA may target ion channels and other proteins independently of CB1 or CB2 receptor activation to elicit its effects. Indeed, several members in the potassium channel super family may interact with cannabinoids to mediate non-CB1, non-CB2 receptor-dependent actions of these lipid messengers. In the following text, I will summarize findings supporting direct modulatory actions of the endocannabinoid AEA on potassium channels and on a few non-potassium channels as examples from other ion channel families . The concentrations of cannabinoids shown in both Tables 1 and 2 represent EC50, IC50, or the range of concentrations being examined in individual studies. Large-conductance, calcium-activated potassium channels are present in most regions of the mammalian brain and in hormone-secreting cells where they modulate neurotransmitter and hormone release through co-localizing with voltagegated calcium channels. In addition, BK channels are expressed in vascular smooth muscle cells where they contribute to the regulation of vascular contractile tone. Moreover, BK channels may participate in the anticonvulsant and vasorelaxant effects of cannabinoids and mediate cannabinoid-induced peripheral analgesia and firing-suppressing effects in primary sensory afferents after nerve injury. Indeed, blocking BK channels reverses the firing-suppression effect of CB receptor agonists and the CB receptor agonist-induced peripheral analgesia. With regard to the mechanism underlying cannabinoid-induced modulation of BK channels, it has been reported that the whole-cell current of BK channels acquired in both transfected human embryonic kidney 293 and native aortic cells is potentiated by the endocannabinoid AEA as well as methanandamide , a synthetic, metabolically stable analog of AEA. The potentiation of BK currents by AEA or methAEA is gradual,rolling greenhouse benches taking around 6–8 minutes to develop a peak response. Notably, the BK-potentiating effect of methAEA is unaffected by a potent CB1 receptor antagonist AM251 or by pertussis toxin that prevents activation of Gi/o protein-coupled receptors, thus excluding an involvement of canonical CB1/CB2 receptors. These results suggest that, by activating BK channels in vascular smooth muscle independently of CB1 and CB2 receptors, endocannabinoids may hyperpolarize membrane potential, reduce cell excitability, and consequently elicit vasodilation, providing neuroprotection after an ischemic stroke and/or suppressing excess activity of vascular smooth muscle tissues. However, the BK channel may not be a direct target of AEA/ methAEA, as methAEA only enhances BK channel activity in whole-cell and cell-attached patch configurations but not in excised inside-out membrane patches. On the other hand, Bondarenko et al. have demonstrated that AEA concentration dependently facilitates single BK channel activity in cell-free, inside-out patches obtained from human endothelial-derived EA.hy926 cells within a physiological Ca2+ range, which suggests that AEA directly interacts with and thereby modifies BK channel activity to induce endothelium dependent vasorelaxation.N-arachidonoyl glycine , an endogenous lipoamino acid structurally and metabolically related to the endocannabinoid AEA, also exhibits analgesic, anti-inflammatory, and proinflammatory-resolving properties. NAgly has been shown to activate BK channels in excised inside out and outside-out patches obtained from human endothelial-derived EA.hy926 cells, and to cause BK channel blocker-sensitive membrane hyperpolarization in in situ mouse aortic endothelium, a critical event to initiate endothelium-dependent vasorelaxation.
This study identifies BK channels as cellular sensors for cannabinoids in in vitro and in situ endothelial cells, an effect that does not require activation of CB1/CB2 receptors or GPR18 , suggesting that NAgly initiates a CB1/CB2 receptor- and GPR18-independent activation of endothelial BK channels, which might contribute to vasodilation to cannabinoids. Interestingly, the action of cannabinoids and cannabinoid-like compounds on endothelial cells is accompanied or at least partially underpinned by modulation of cholesterol level in caveolae, as the stimulatory response of endothelial BK channels to NAgly or to AEA is prevented following cholesterol depletion with methyl-β-cyclodextrin.Voltage-gated potassium channels shape the action potential by controlling its repolarization phase and determine the membrane potential and duration of the interspike interval. In general, delayed rectifier Kv channels function to keep single action potentials short and to permit high frequency trains of action potentials, whereas rapidly inactivating A-type potassium channels space repetitive responses and help a cell fire at low frequencies.A-type currents are calcium-independent Kv currents that undergo rapid activation and inactivation. A-type currents have been identified and characterized in neuronal, cardiac, vascular, genitourinary, and gastrointestinal smooth muscle cells. In the heart, potassium channels play important roles in determining the firing frequency in sinus node pacemaker cells as well as resting potential and the shape and duration of action potentials in cardiomyocytes; A-type currents present in atrial and ventricular myocytes are referred to as “transient” outward current . A complex formed by Kv4.2, Kv4.3, and KChIP2 may underlie the fast transient outward current in cardiac muscle, while Kv1.4 may underlie a slower transient outward current. Endocannabinoids are involved in the regulation of cardiovascular function. It has been demonstrated by Amorós et al. that the whole-cell current of cloned human cardiac Kv4.3/KChIP2 channels expressed in stably transfected Chinese hamster ovary cells is inhibited directly by AEA, 2-AG, and methAEA in a concentration-dependent fashion . The inhibition is accompanied by accelerated inactivation and a hyperpolarization shift in the voltage dependence of inactivation. Moreover, these inhibitory effects of endocannabinoids on IKv4.3 are not mediated by activation of CB1/CB2 receptors or by modifications of the lipid order and microviscosity of the cell membrane; furthermore, the putative AEA-interacting site has been suggested to reside in the Kv4.3 α- subunit at its extracellular surface, as AEA and methAEA only block Kv4.3 channels when administered from the outside. Human cardiac Kv4.3 channels thus represents a novel molecular target for AEA, 2-AG and cannabinoid analogues; the potent, direct inhibition of human cardiac IKv4.3 and Ito, fast by AEA and 2-AG would increase the height and prolong the plateau duration of human cardiac action potential, altering human cardiac electrical activity. Potassium channels are the basis for the change in action potential configuration in response to variation in heart rate and they are highly regulated. Using isolated rat ventricular myocytes as a cell model, Li et al. investigated the electrophysiological effects of AEA on K+ currents and reported that AEA concentration-dependently decreases the rapidly activating and inactivating transient outward current Ito. Kinetically, AEA shifts the steady state inactivation curve of Ito to the left and the recovery curve of Ito to the right, suggesting that AEA accelerates the voltage-dependent steady-state inactivation of Ito and suppresses Ito recovery from inactivation. The maximal effect of AEA on Ito develops slowly but can be measured within 8 min of initial exposure during continuous drug application. Neither CB1 receptor antagonist AM251 nor CB2 receptor antagonist AM630 can abolish the inhibitory effect of AEA on Ito, which further suggests that AEA reduces Ito through a non-CB1 and non-CB2 receptor-mediated mechanism. In contrast, AEA exerts no effect on steady-state outward K+ current and the inward rectifier current in rat ventricular myocytes. Cardiac Ito channels represent an important target of class III antiarrhythmic drugs, owing to their critical role in defining resting membrane potential, heart rate, and action potential shape and duration in cardiac sinus node cells and cardiac myocytes. Inhibition of Ito channels by AEA may therefore account for, at least in part, the antiarrhythmic action of AEA. In substantia nigra pars compacta dopaminergic neurons, the fast inactivating A-type K+ current is mediated by Kv4.3 channels co-assembled with the accessory subunits KChIP3.1. Gantz and Bean reported that at 37°C, IA in isolated mouse midbrain dopaminergic neurons is inhibited by 2-AG , the dominant endocannabinoid in the brain, in a concentration-dependent manner; moreover, this acute inhibitory effect of 2-AG on IA does not require CB receptor activation or G protein signaling.