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 G beta gamma 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 G beta gamma, but a G beta gamma-independent component has also been envisaged. We investigated G beta gamma dependence of the basal GIRK activity (A(GIRK,basal)) quantitatively, by titrated expression of G beta gamma scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used G beta gamma scavenger, myristoylated C terminus of beta-adrenergic kinase (m-c beta ARK), reduced A(GIRK,basal) by 70 - 80% and eliminated the acetylcholine-evoked current (I-ACh). However, we found that m-c beta ARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed G beta gamma 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 G beta gamma and G alpha in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of G beta gamma and its constitutive association with GIRK may underlie the excessively high AGIRK, basal observed at high expression levels of GIRK. Only 10 - 15% of A(GIRK, basal) persisted upon expression of both m-phosducin and c beta ARK. These results demonstrate that a major part of I-basal is G beta gamma-dependent at all levels of channel expression, and only a small fraction (< 10%) may be G beta gamma-independent.