βL–βM loop in the C‐terminal domain of G protein‐activated inwardly rectifying K⁺ channels is important for G_βγ subunit activation

Abstract

The activity of G protein-activated inwardly rectifying K + channels (GIRK or Kir3) is important for regulating membrane excitability in neuronal, cardiac and endocrine cells. Although G βγ subunits are known to bind the N-and C-termini of GIRK channels, the mechanism underlying G βγ activation of GIRK is not well understood. Here, we used chimeras and point mutants constructed from GIRK2 and IRK1, a G protein-insensitive inward rectifier, to determine the region within GIRK2 important for G βγ binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C-terminal domain of GIRK2, GIRK2 L344E and GIRK2 G347H , that exhibited decreased carbacholactivated currents but significantly enhanced basal currents with coexpression of G βγ subunits. Combining the two mutations (GIRK2 EH ) led to a more severe reduction in carbachol-activated and G βγ -stimulated currents. Ethanol-activated currents were normal, however, suggesting that G protein-independent gating was unaffected by the mutations. Both GIRK2 L344E and GIRK2 EH also showed reduced carbachol activation and normal ethanol activation when expressed in HEK-293T cells. Using epitope-tagged channels expressed in HEK-293T cells, immunocytochemistry showed that G βγ -impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced G βγ activation. In vitro G βγ binding assays revealed an ∼60% decrease in G βγ binding to the C-terminal domain of GIRK2 L344E but no statistical change with GIRK2 EH or GIRK2 G347H , though both mutants exhibited G βγ -impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in G βγ activation of GIRK2 channels. Based on the 1.8Å structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the βL-βM loop, which is situated away from the pore and near the N-terminal domain. The results are discussed in terms of a model for activation in which G βγ alters the interaction between the βL-βM loop and the N-terminal domain.

Molecular mechanism underlying ethanol activation of G-protein–gated inwardly rectifying potassium channels

“…Previous reports suggested a difference in alcohol and receptor activation mechanisms (4,5). In support of this finding, the rate of MTS-HE-dependent inhibition of basal currents in L344C, part of the Gβγ binding site in the βL-βM loop (28)(29)(30), was dependent on Gβγ levels, whereas MTS-HE activation of L257C channels was unaffected by varying Gβγ levels. However, alcohol-dependent (this study), Gβγ-dependent (19), and Na +z -dependent (25,(40)(41)(42) activation all involve changes in PIP 2 -GIRK affinity, suggesting a convergence of Fig.…”

“…Receptor-dependent Gβγ activation, however, relies on direct protein-protein interaction between GIRKs and G proteins (21,(28)(29)(30). Previous reports suggested a difference in alcohol and receptor activation mechanisms (4,5).…”

Selective Gαi Subunits as Novel Direct Activators of Transient Receptor Potential Canonical (TRPC)4 and TRPC5 Channels

“…1-4 indicate that activation of G␣ i subunits by GPCRs is the primary mechanism for activating TRPC4 and TRPC5. This raised the question of whether activation of the channels requires direct interaction with the G␣ i subunits, as was shown for other channels regulated by G␣ (36) and G␤␥ (33,37) subunits. To address this question, we identified the TRPC4 and TRPC5 domain that might interact with the G␣ i subunits.…”

Gβγ-dependent and Gβγ-independent Basal Activity of G Protein-activated K+ Channels

“…Later works suggested that an intact putative site underlying A GIRK,basal is also crucial for agonist-evoked activity (58) and that a residue implicated in low affinity G␤␥ binding (57) may be involved in channel gating rather than G␤␥ binding (21,59). Thus, the issue of two types of G␤␥-binding site remains unresolved.…”