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βL–βM loop in the C‐terminal domain of G protein‐activated inwardly rectifying K + channels is important for G βγ subunit activation
Author(s) -
Finley Melissa,
Arrabit Christine,
Fowler Catherine,
Suen Ka Fai,
Slesinger Paul A.
Publication year - 2004
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2003.056101
Subject(s) - g protein coupled inwardly rectifying potassium channel , g protein , heterotrimeric g protein , inward rectifier potassium ion channel , chemistry , hek 293 cells , microbiology and biotechnology , xenopus , gq alpha subunit , patch clamp , biophysics , biology , biochemistry , ion channel , receptor , gene
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 carbachol‐activated 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 1.8 Å 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.