Gβγ-dependent and Gβγ-independent basal activity of G protein-activated K⁺ channels

Cardiac and neuronal G protein-activated K⁺ channels (GIRK; Kir3) open following the binding of Gβγ subunits, released from G_i/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free Gβγ, but a Gβγ-independent component has also been envisaged. We investigated Gβγ dependence of the basal GIRK activity (A_GIRK,basal) quantitatively, by titrated expression of Gβγ scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used Gβγ scavenger, myristoylated C terminus of β-adrenergic kinase (m-cβARK), reduced A_GIRK,basal by 70-80% and eliminated the acetylcholine-evoked current (I_ACh). However, we found that m-cβARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed Gβγ scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents, m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of A_GIRK,basal by up to 90%. Expression of GIRK was accompanied by an increase in the level of Gβγ and Gα in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of Gβγ and its constitutive association with GIRK may underlie the excessively high A_GIRK,basal observed at high expression levels of GIRK. Only 10-15% of A_GIRK,basal persisted upon expression of both m-phosducin and cβARK. These results demonstrate that a major part of I_basal is Gβγ-dependent at all levels of channel expression, and only a small fraction (<10%) may be Gβγ- independent.