Differential effects of genetically-encoded G_(βγ) scavengers on receptor-activated and basal Kir3.1/Kir3.4 channel current in rat atrial myocytes

摘要

Opening of G-protein-activated inward-rectifying K~+ (GIRK, Kir3) channels is regulated by interaction with βγ-subunits of Pertussis-toxin-sensitive G proteins upon activation of appropriate GPCRs. In atrial and neuronal cells agonist-independent activity (I_(basal)) contributes to the background K~+ conductance, important for stabilizing resting potential. Data obtained from the Kir3 signaling pathway reconstituted in Xenopus oocytes suggest that Ibasal requires free G_(βγ). In cellswith intrinsic expression of Kir3 channels this issue has been scarcely addressed experimentally. Two G_(βγ)-binding proteins (myristoylated phosducin-mPhos-and G_(αi1)) were expressed in atrial myocytes using adenoviral gene transfer, to interrupt G_(βγ)-signaling. Agonist-induced and basal currents were recorded using whole cell voltage-clamp. Expression of mPhos and G_(αi1) reduced activation of Kir3 current via muscarinic M_2 receptors (IK(ACh)). Inhibition of I_(K(ACh)) by mPhos consisted of an irreversible component and an agonist-dependent reversible component. Reduction in density of I_(K(ACh)) by overexpressed Gαi1, in contrast to mPhos, was paralleled by substantial slowing of activation, suggesting a reduction in density of functional M_2 receptors, rather than G_(βγ)-scavenging as underlying mechanism. In line with this notion, current density and activation kinetics were rescued by fusing the αi1-subunit to an Adenosine A_1 receptor. NeithermPhos nor G_(αi1) had a significant effect on I_(basal), defined by the inhibitory peptide tertiapin-Q. These data demonstrate that basal Kir3 current in a native environment is unrelated to G-protein signaling or agonist-independent free G_(βγ). Moreover, our results illustrate the importance of physiological expression levels of the signaling components in shaping key parameters of the response to an agonist.