| Autor: |
Finley, Melissa, Arrabit, Christine, Fowler, Catherine, Ka Fai Suen, Catherine, Slesinger, Paul A. |
| Předmět: |
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| Zdroj: |
Journal of Physiology; Mar2004, Vol. 555 Issue 3, p643-657, 15p, 1 Black and White Photograph, 6 Graphs |
| Abstrakt: |
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 Gβγ subunits are known to bind the N- and C-termini of GIRK channels, the mechanism underlying Gβγ 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 Gβγ binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C-terminal domain of GIRK2, GIRK2L344E and GIRK2G347H, that exhibited decreased carbachol-activated currents but significantly enhanced basal currents with coexpression of Gβγ subunits. Combining the two mutations (GIRK2EH) led to a more severe reduction in carbachol-activated and Gβγ-stimulated currents. Ethanol-activated currents were normal, however, suggesting that G. protein-independent gating was unaffected by the mutations. Both GIRK2L344E and GIRK2EH 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 Gβγ-impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced Gβγ activation. In vitro Gβγ binding assays revealed an ∼ 6O% decrease in Gβγ binding to the C-terminal domain of GIRK2L344E but no statistical change with GIRK2EH or GIRK2G347H, though both mutants exhibited Gβγ-impaired activation. Together, these... [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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