Mapping the Gβγ-binding sites in GIRK1 and GIRK2 subunits of the G protein-activated K+ channel

Tatiana Ivanina, Ida Rishal, Dalia Varon, Carmen Müllner, Bibiane Frohnwieser-Steinecke, Wolfgang Schreibmayer, Carmen W. Dessauer, Nathan Dascal*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

G protein-activated K+ channels (Kir3 or GIRK) are activated by direct binding of Gβγ. The binding sites of Gβγ in the ubiquitous GIRK1 (Kir3.1) subunit have not been unequivocally charted, and in the neuronal GIRK2 (Kir3.2) subunit the binding of Gβγ has not been studied. We verified and extended the map of Gβγ-binding sites in GIRK1 by using two approaches: direct binding of Gβγ to fragments of GIRK subunits (pull down), and competition of these fragments with the Gαi1 subunit for binding to Gβγ. We also mapped the Gβγ-binding sites in GIRK2. In both subunits, the N terminus binds Gβγ. In the C terminus, the Gβγ-binding sites in the two subunits are not identical; GIRK1, but not GIRK2, has a previously unrecognized Gβγ-interacting segments in the first half of the C terminus. The main C-terminal Gβγ-binding segment found in both subunits is located approximately between amino acids 320 and 409 (by GIRK1 count). Mutation of C-terminal leucines 262 or 333 in GIRK1, recognized previously as crucial for Gβγ regulation of the channel, and of the corresponding leucines 273 and 344 in GIRK2 dramatically altered the properties of K+ currents via GIRK1/GIRK2 channels expressed in Xenopus oocytes but did not appreciably reduce the binding of Gβγ to the corresponding fusion proteins, indicating that these residues are mainly important for the regulation of Gβγ-induced changes in channel gating rather than Gβγ binding.

Original languageEnglish
Pages (from-to)29174-29183
Number of pages10
JournalJournal of Biological Chemistry
Volume278
Issue number31
DOIs
StatePublished - 1 Aug 2003

Funding

FundersFunder number
National Institute of General Medical SciencesR01GM060419

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