Abstract
G protein-gated inwardly rectifying K+ (GIRK) channels are known to be activated by direct binding of Gβγ released from Gi/o protein, and inhibited by stimulation of coexpressed Gq-coupled receptors. Inhibitory mechanisms of the GIRK channels are thought to be due to phosphorylation by PKC and/or depeletion of PIP2. To better understand the inhibitory mechanisms, we analysed the GIRK current expressed in Xenopus oocytes under two-electrode voltage clamp, focusing on the turning -on and -off phases upon application or removal of the ligands of the coexpressed Gi/o-coupled receptors. The time constants, τon and τoff, obtained by fitting the -on and -off phases with single exponential functions, are parameters which reflect Gβγ binding to or dissociation from the GIRK channels. By pre-stimulating Gq-coupled receptors coexpressed with wild-type GIRK channels, we observed reductions of the basal and ligand induced current amplitudes, an increase in τon and a decrease in τoff. As PIP2 is abundant in oocytes, we expected the effect of PIP2 depletion to be minor, and focused on the phosphorylation by PKC. When PMA, a direct activator of PKC, was applied in advance, similar changes were observed. These changes were not observed in the GIRK1(S185A)/GIRK2(S196A) mutant whose PKC-phosphorylation motifs are mutated. These results suggest that the affinity of GIRK channel to Gβγ is decreased by phosphorylation of these sites by PKC, resulting in speed-up of turning on, slow down of turning off and reduction of current amplitude. [Jpn J Physiol 55 Suppl:S125 (2005)]
