The changes in A-type potassium channel gating caused by 2-AG include a shift of the voltage-dependent activation of IA in the depolarizing direction, acceleration of the inactivation kinetics, and reduction of the maximal current for large depolarizations. The IA–inhibiting effect of 2-AG likely results from a direct action on the channel through a membrane lipid interaction; specifically, binding of 2-AG may modify the interaction of the voltage-sensing S4-S5 region of the channel with the S6 region that controls channel opening and closing. It is thus suggested that by modulating IA, 2-AG and related lipid signaling molecules can directly tune neuronal excitability in a cellautonomous manner. 2) Delayed rectifiers: Besides exerting inhibitory effects on fast–inactivating potassium channels as described above, endocannabinoids also modulate the activity of classical delayed rectifier-type potassium channels. Classical delayed rectifiers like Kv1.2 , Kv2.1, and Kv3.1 do not show inactivation in the millisecond time scale; functionally, delayed rectifiers terminate action potentials, restore the dominant potassium permeability of the resting membrane potential, and shape the action potential. AEA has been shown to inhibit Shaker-related Kv1.2 channels; specifically, via accelerating the inactivation rate of Kv1.2, externally applied AEA concentration-dependently reduces Kv1.2 channel currents recorded at whole-cell and outside-out patch modes in fibroblasts stably transfected with Kv1.2 cDNA, whereas intracellularly dialyzed AEA is without effect. This inhibitory effect of AEA on Kv1.2 channels does not require activation of CB1 receptors or G protein signaling since neither CB1 receptor antagonists nor pertussis toxin could prevent the effect.
Moreover,rolling grow table externally applied Δ9 -THC, the major psychotropic constituent of cannabis, is capable of mimicking the inhibitory action of AEA on the Kv1.2 channel. Poling et al. thus propose that an acceptor site on the extracellular side of the Kv1.2 channel recognizes AEA and other cannabinoid-like molecules. In vascular smooth muscle cells, modulation of potassium channel activity plays an essential role in regulating membrane potential, which in turn influences the open probability of vascular CaV channels, the contractile tone of vascular smooth muscle, and blood flow . External application of AEA has been reported to concentration dependently reduce the delayed rectifier K+ current acquired in the whole-cell mode from freshly dissociated rat aortic smooth muscle cells in a CB1 receptor-independent manner. In addition, methAEA and WIN 55,212–2 elicit similar inhibition of delayed rectifier K+ current, and these effects are also CB receptor independent. Van den Bossche and Vanheel thus concluded that cannabinoids likely bind to an external site on or near the delayed rectifier Kv channel of the aortic vascular smooth muscle cells to modulate the channel activity. Intriguingly, it appears that AEA may inhibit delayed rectifiers in different tissues via a similar mechanism, i.e., acting from the extracellular side of the membrane . It has been demonstrated that in both primary cultured rat cortical astrocytes and astroglial cells in cortical slices, low micromolar concentrations of AEA promote a strong reduction of the delayed rectifier outward K+ current. Pharmacological blockade experiments further uncovered the AEA-induced inhibition is independent of CB1 receptor activation, AEA metabolism, and Ca2+ signaling. The delayed rectifier-inhibiting effect of AEA in astrocytes is mediated by its interaction with the extracellular leaflet of the plasma membrane based on the observation that only extracellularly, but not intracellularly, applied bovine serum albumin recovers the delayed rectifier K+ current suppressed by AEA.
Moreover, the inhibitory effect of AEA on astrocyte delayed rectifier potassium channels does not involve an interaction of AEA with lipid rafts and/or caveolae as cholesterol-extracting agents fail to abrogate the AEA effect. Collectively, the findings made by Vignali et al. suggest that AEA may stabilize the closed state of the delayed rectifier potassium channel by binding to hydrophobic determinants of the protein complex. Their findings support that endocannabinoids may effectuate their modulation of CNS function through regulating potassium channel-mediated homeostatic function of the astroglial syncytium, which might explain some non-neuronal effects of the endocannabinoid system . In the pancreas, Kv channels contribute to the regulation of insulin secretion by controlling the repolarization of pancreatic β-cell action potential. The delayed rectifier is thought to be the dominant Kv current of β-cells, and hence has received much attention as potential therapeutic targets for type 2 diabetes. It is noteworthy that delayed rectifier Kv channels in β-cells are also modulated by endocannabinoids. Spivak et al. reported that 2-AG concentration dependently inhibits the whole-cell current of delayed rectifier potassium channels in the mouse insulinoma cell line R7T1, with an IC50 of 20 μM; moreover, the inhibitory effect of 2-AG on β–cell delayed rectifier K+ current is CB receptor independent. The predominant delayed rectifier potassium channel in murine β-cells is the Kv2.1 type; moreover, delayed rectifiers from Kv1 , Kv2, and Kv3 subfamilies are also present in murine β-cells. In addition, Kv1.5 is detected in human insulinoma cells and is highly expressed in human islets. It is possible that multiple distinct Kv channels comprise the delayed rectifier current of the human ß- cell . In the human heart, Kv1.5 channels underlie the ultra-rapidly activating delayed rectifier K+ current that is critical for determining the height and duration of the human atrial action potential and a potential target for treating atrial arrhythmias. It has been demonstrated by Barana et al. that both AEA and 2-AG exhibit a high potency to inhibit human Kv1.5 current acquired in stably transfected mouse fibroblasts, an inhibitory effect that is independent of CB receptor activation and changes in the order and microviscosity of the membrane; in other words, the potencies of blockade are not related to the liposolubility of the compounds.
Endocannabinoidinduced block of human cardiac Kv1.5 channels appears exclusively when endocannabinoids are applied at the external surface of the cell membrane; furthermore, the blockade by AEA or 2-AG is reduced by mutation of R487 located in the external vestibule-entryway, a residue that determines Kv1.5 sensitivity to external tetraethylammonium . AEA also inhibits atrial end pulse sustained K+ current in human atrial myocytes which is mainly carried by Kv1.5 channels, and it significantly prolongs the duration of action potentials recorded in mouse left atria. These findings thus support that endocannabinoids block human cardiac Kv1.5 channels via specific interaction at the extracellular TEA binding site of the channel, a mechanism by which the endocannabinoids regulate the shape of atrial action potentials. Moreno-Galindo et al. examined AEA modulation of cloned Kv1.5 channels expressed in transfected HEK293 cells and showed that AEA exerts high-potency block of Kv1.5 channel currents in a CB receptor independent manner from the cytoplasmic membrane surface,cannabis grow equipment consistent with open-channel block. The postulated binding site for AEA in the study by Moreno-Galindo et al. is located on the S6 domain that lines the channel vestibule, which, however, is distinct from the extracellularly located interaction site suggested by Barana et al.. Specifically, Moreno-Galindo et al. identified Val505 and Ile508 within the S6 domain of Kv1.5 , two residues facing toward the central cavity and constituting a motif that forms a hydrophobic ring around the ion conduction pathway, as AEA interacting sites. Moreno-Galindo et al. thus suggest that the hydrophobic ring motif may be a critical determinant of CB receptor-independent AEA modulation in other K+ channel families. Studies reviewed above support that the endocannabinoid AEA may directly interact with delayed rectifier Kv channels and thereby modulate the channel function in a CB1/CB2 receptor independent manner. Interestingly, observations made on cloned Kv channels expressed in Xenopus oocytes have revealed that membrane lipids such as PIP2, by removing fast inactivation, can convert A-type potassium channels into delayed rectifiers, whereas AEA and arachidonic acid, by introducing fast inactivation, can convert non-inactivating delayed rectifiers into rapidly inactivating A-type potassium channels. These findings thus imply that AEA may control the coding properties of neurons and synapses beyond the characteristics set by the expression profile of Kv channel protein subunits.In addition to BK and Kv channels, endocannabinoids also modulate two-pore domain potassium channels. TWIK-related acid-sensitive potassium channel 1 , a member in the K2P channel subfamily, encodes an acid- and anesthetic-sensitive background K+ current, which sets the resting membrane potential of both cerebellar granule neurons and somatic motoneurons, and may contribute to anesthetic induced immobilization.
It has been demonstrated by Maingret et al. that, unlike other K2P channels, TASK-1 expressed in COS-7, CHO or HEK293 cells is directly blocked by submicromolar concentrations of AEA, an effect independent of both CB1 and CB2 receptors and G proteins. The inhibition of TASK-1 by AEA is specific, not mimicked by 2-AG, another endocannabinoid, or by Δ9 -THC, the major psychoactive compound in cannabis; additionally, AEA hydrolysis is not involved, as the non-hydrolysable analogue methAEA is similarly effective. These findings suggest that TASK-1 constitutes a novel, sensitive molecular target of AEA. It is recognized that cannabinoids including AEA profoundly affect locomotion, exerting a dose-related biphasic effect ; direct modulation of TASK-1 by low doses of AEA might thus account for some of the biphasic, CB1 receptor-independent effects observed with AEA on locomotion.The role played by CB receptors in regulating energy balance is well established. Endocannabinoid lipids are known to exert orexigenic effects via activation of central cannabinoid CB1 and CB2 receptors; in addition, the peripherally produced endocannabinoids also act as local regulators of insulin secretion through pancreatic β-cell CB receptor-mediated elevation in Ca2+ levels. Beyond a physiological role in regulating energy balance, the cannabinoid transduction cascade may have a pathophysiological function to stimulate insulin-dependent lipid deposition through enhanced insulin output in response to increased levels of peripheral 2-AG, as seen in obesity, which implies that a dysregulated endocannabinoid system in the adipocytes and β- cells likely contributes to hyperlipidemia, hypoadiponectinemia, and hyperinsulinemia in obesity. The ATP-sensitive potassium channel, a member in the inwardly rectifying potassium channel subfamily, functions as a high fidelity metabolic sensor that couples intracellular metabolic state to membrane excitability, serving a homeostatic role ranging from blood glucose regulation to cardioprotection. KATP channels in pancreatic β-cells regulate insulin secretion in response to plasma glucose levels, via regulating membrane potential and thereby the activity of CaV channels, intracellular Ca2+ levels, and Ca2+-dependent exocytosis of insulin. Interestingly, β-cell KATP channels have been shown to be modulated by endocannabinoids. Spivak et al. reported that single-channel KATP currents acquired at 2 mM glucose in the inside-out patch configuration in R7T1 cells, a mouse insulinoma β-cell line, are concentration dependently inhibited by 2-AG applied from the cytosolic side . CB1 receptors are expressed in murine β-cells; however, the CB1 receptor antagonist AM251 does not affect 2-AG’s inhibitory action on KATP channel current, indicating that CB1 receptors do not mediate the effect. The direct blockade of the KATP channel by 2-AG at low glucose concentrations would depolarize the β-cell and results in stimulation of insulin secretion; it is suggested that 2-AG may increase insulin secretion in a manner similar to that of the sulphonylureas by directly interacting with the KATP channel. However, a role of AEA on the function of pancreatic β-cell KATP channels has yet to be explored. Endogenous KATP channels in follicle-enclosed oocytes from Xenopus laevis are subject to modulation by gonadotropins and may play important roles in oocyte maturation and hormonal regulation of oocyte development. The effect of endocannabinoids on cromakalim -activated KATP currents has been examined in follicular oocytes using two-electrode voltage-clamp recordings. AEA inhibits cromakalim-activated KATP currents in a noncompetitive manner, with an IC50 of 8.1 μM; the inhibitory effect of AEA on cromakalim-induced KATP currents is independent of CB receptors and of Gi/o-coupled receptors, as manifested by the ineffectiveness of CB receptor antagonists and pertussis toxin, respectively, to prevent AEA-induced block. Furthermore, inhibitors for AEA’s degradative enzymes amidohydrolase and cyclooxygenase fail to affect the blockade of cromakalim-induced KATP currents caused by AEA, indicating that the effect of AEA is not mediated by its metabolic products. Evidence provide by Oz et al. thus suggests that AEA may modulate the hormonal maturation process in Xenopus oocytes by modulating KATP channel activity. AEA is involved in the regulation of cardiovascular function. In addition to Kv channels in ventricular myocytes and vascular smooth muscle cells , myocardial KATP channels are also functionally modulated by AEA. Contrary to the observations made in pancreatic β–cells and follicle-enclosed oocytes where endocannabinoids suppress native KATP channel function, Li et al. reported that in isolated rat ventricular myocytes, AEA increases whole-cell KATP currents induced by dinitrophenol, a mitochondrial uncoupler, in a concentration dependent manner. Furthermore, the stimulatory effect of AEA on ventricular KATP currents is reduced by pretreatment of cells with the CB2 receptor antagonist AM630, whereas the CB1 receptor antagonist AM251 has no effect.