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

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Abstract
We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca2+-activated K+ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the α-subunit of the BK channel (BK-α), both α- and β1-subunits (BK-αβ1), or both α- and β4-subunits (BK-αβ4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-αβ1 within a similar concentration range. Because methAEA could potentiate BK-α, BK-αβ1, and BK-αβ4 with similar efficacy, the β-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-α and HEK-BK-αβ1 did not change intracellular Ca2+ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB2 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.