Activation of large-conductance, Ca²⁺-activated K⁺ channels by cannabinoids

Abstract

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca 2ϩ -activated K ϩ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the ␣-subunit of the BK channel (BK-␣), both ␣-and ␤ 1-subunits (BK-␣␤1), or both ␣-and ␤4-subunits (BK-␣␤4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-␣␤ 1 within a similar concentration range. Because methAEA could potentiate BK-␣, BK-␣␤ 1, and BK-␣␤4 with similar efficacy, the ␤-subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK-␣ and HEK-BK-␣␤ 1 did not change intracellular Ca 2ϩ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB 1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB 2 agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed.anandamide; channel opener LARGE-CONDUCTANCE Ca 2ϩ -activated K ϩ (BK) channels consist of channel-forming ␣-subunit and accessory ␤-subunits (␤ 1 -␤ 4 ) arranged in tetramers (24). Each ␤-subunit interacts with the NH 2 -terminal region of the ␣-subunit and regulates the activity of the ␣-subunit by changing Ca 2ϩ and voltage sensitivity and/or channel kinetics (10, 11). Although only one major type of ␣-subunit with splice variants has been found, several subtypes of ␤-subunits whose ␤ 1 -and ␤ 4 -isoforms are abundantly expressed in smooth muscle and central nervous system (CNS), respectively, have been cloned and may be responsible for the tissue-specific characteristics of BK channels (4,36,44). Synthetic compounds such as NS-1619 and BMS-204352 are activators of the BK-␣ subunit, whereas dehydrosoyasaponin I (14), 17␤-estradiol (37, 3), and tamoxifen (9) act on the BK ␤ 1 -subunit. Agents that enhance BK channel activity (BK channel openers) may be effective in protecting neurons from damage after an ischemic stroke and/or in suppressing excess activity of smooth muscle tissues (23, 35). 17␤-Estradiol (37), arachidonic acid (AA) (21), epoxyeicosatrienoic acids (EETs) (13), and dihydroxyeicosatrienoic acids (25) may be endogenous BK channel openers, and some transmitters and hormones also can enhance BK channel ...

The Endogenous Cannabinoid Anandamide Inhibits Cromakalim-Activated K+Currents in Follicle-EnclosedXenopusOocytes

“…In agreement with our results, several earlier studies indicated that different types K ϩ channels are modulated by anandamide in a cannabinoid receptor-independent manner (Poling et al, 1996;Van den Bossche and Vanheel, 2000;Maingret et al, 2001;Oliver et al, 2004;Sade et al, 2006). Anandamide belongs to a class of signaling lipids consisting of amides of long-chain polyunsaturated fatty acids (Howlett et al, 2002).…”

“…For example, in excitable cells such as neurons, the activation of cannabinoid receptors by anandamide suppresses the presynaptic release of various neurotransmitters by inhibiting the function of voltage-dependent Ca 2ϩ channels (Howlett et al, 2002). Other studies have shown that anandamide modulates K ϩ channels (Poling et al, 1996;Van den Bossche and Vanheel, 2000;Maingret et al, 2001;Oliver et al, 2004;Sade et al, 2006). ATP-sensitive potassium (K ATP ) channels form an important link between metabolic state and cell excitability.…”

International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid Receptors and Their Ligands: Beyond CB1and CB2

“…Results obtained in experiments with rat isolated coronary arteries also suggest that such activation can be produced by both anandamide and R-(ϩ)-methanandamide at concentrations of 0.3 to 3 M, although not by JWH-133 at 1 M (Sade et al, 2006).…”

Endocannabinoids and Their Pharmacological Actions

“…Thus, it has been found that this endocannabinoid induces such an increase in BK-αβ 1 channels expressed by HEK-293 cells, at 0.3, 1 and 3 μM (Sade et al 2006), in human BK K Ca channels expressed by HEK-293 cells, with an EC 50 value of 4.8 μM (Godlewski et al 2009), and in small conductance calcium-activated SK K Ca channels expressed naturally by rat cultured hippocampal neurons, at 1-100 nM (Wang et al 2011). Evidence was obtained as well in some of these investigations that anandamide did not increase K þ currents in BK or SK K Ca channels by acting on CB 1 or CB 2 receptors or indeed on TRPV1 channels (Sade et al 2006;Wang et al 2011). There has been a report too that outward K þ currents in BK K Ca channels expressed by HEK-293 cells are unaffected by 2-arachidonoylglycerol (10 μM) and that they are decreased by virodhamine with an IC 50 value of 447 nM (Godlewski et al 2009).…”