Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Pflugers Arch
2008 Mar 01;4556:1017-24. doi: 10.1007/s00424-007-0358-4.
Show Gene links
Show Anatomy links
Subunit stoichiometry of heterologously expressed G-protein activated inwardly rectifying potassium channels analysed by fluorescence intensity ratio measurement.
Grasser E
,
Steinecker B
,
Ahammer H
,
Schreibmayer W
.
???displayArticle.abstract???
The Xenopus laevis oocyte expression system offers the unique opportunity to heterologously express many proteins simultaneously and to control the expression level for every protein individually. By using the expression of fusion constructs of variants of the green fluorescence protein (eCFP, eGFP and eYFP) with GIRK1 and GIRK4 subunits and measuring the respective fluorescence intensity ratios (FIRs) of the expressed proteins by confocal laser scan microscopy, we were able to measure the amount of each of the individual subunits expressed. At equal amounts of injected RNAs encoding GIRK1 and GIRK4, we found that approximately 2.2 GIRK4 subunits per 1 GIRK1 subunit appeared at the surface of the oocyte, suggesting the coexistence of homooligomeric GIRK4 complexes with heterooligomeric GIRK1/GIRK4 complexes. Interestingly, when the ratio of injected RNA is increased in favour of GIRK1, the subunit stoichiometry changes accordingly until, at a RNA ratio of 25:1 (GIRK1/GIRK4), the subunit stoichiometry is shifted towards a protein complex with 3:1 stoichiometry (GIRK1/GIRK4). In parallel, the amount of GIRK1 protein appearing at the surface gets greatly reduced, supporting previous studies that showed that the GIRK1 subunit needs assembly with GIRK4 for surface localization. By using a genetically encoded marker for the endoplasmic reticulum (ER), we were able to show that the subunit stoichiometry in regions of the ER, which are located directly below the plasma membrane, closely resembles that observed directly at the surface. Generally, our study reveals that the subunit stoichiometry of GIRK1/GIRK4 channels in the Xenopus laevis oocyte expression system depends to a great extent on the molar ratio of the different RNAs injected.
Chan,
Specific regions of heteromeric subunits involved in enhancement of G protein-gated K+ channel activity.
1997, Pubmed,
Xenbase
Chan,
Specific regions of heteromeric subunits involved in enhancement of G protein-gated K+ channel activity.
1997,
Pubmed
,
Xenbase
Corey,
Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh.
1998,
Pubmed
Corey,
Identification of native atrial G-protein-regulated inwardly rectifying K+ (GIRK4) channel homomultimers.
1998,
Pubmed
Dascal,
The use of Xenopus oocytes for the study of ion channels.
1987,
Pubmed
,
Xenbase
Dascal,
Expression and modulation of voltage-gated calcium channels after RNA injection in Xenopus oocytes.
1986,
Pubmed
,
Xenbase
Delling,
The neural cell adhesion molecule regulates cell-surface delivery of G-protein-activated inwardly rectifying potassium channels via lipid rafts.
2002,
Pubmed
,
Xenbase
Fliegel,
Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum.
1989,
Pubmed
Hedin,
Cloning of a Xenopus laevis inwardly rectifying K+ channel subunit that permits GIRK1 expression of IKACh currents in oocytes.
1996,
Pubmed
,
Xenbase
Hofer,
A comparative study of the action of tolperisone on seven different voltage dependent sodium channel isoforms.
2006,
Pubmed
,
Xenbase
Ivanina,
Expression of GIRK (Kir3.1/Kir3.4) channels in mouse fibroblast cells with and without beta1 integrins.
2000,
Pubmed
Kennedy,
GIRK4 confers appropriate processing and cell surface localization to G-protein-gated potassium channels.
1999,
Pubmed
Krapivinsky,
The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K(+)-channel proteins.
1995,
Pubmed
,
Xenbase
Ma,
Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart.
2002,
Pubmed
McPhee,
Evidence for a functional interaction between integrins and G protein-activated inward rectifier K+ channels.
1998,
Pubmed
Mirshahi,
Molecular determinants responsible for differential cellular distribution of G protein-gated inwardly rectifying K+ channels.
2004,
Pubmed
,
Xenbase
Pelham,
The dynamic organisation of the secretory pathway.
1996,
Pubmed
Riven,
Conformational rearrangements associated with the gating of the G protein-coupled potassium channel revealed by FRET microscopy.
2003,
Pubmed
,
Xenbase
Sarac,
Mutation of critical GIRK subunit residues disrupts N- and C-termini association and channel function.
2005,
Pubmed
,
Xenbase
Schreibmayer,
Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes.
1994,
Pubmed
,
Xenbase
Silverman,
Subunit stoichiometry of a heteromultimeric G protein-coupled inward-rectifier K+ channel.
1996,
Pubmed
,
Xenbase
Stevens,
Identification of regions that regulate the expression and activity of G protein-gated inward rectifier K+ channels in Xenopus oocytes.
1997,
Pubmed
,
Xenbase
Tucker,
Muscarine-gated K+ channel: subunit stoichiometry and structural domains essential for G protein stimulation.
1996,
Pubmed
,
Xenbase
Vivaudou,
Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants.
1997,
Pubmed
,
Xenbase
Wickman,
Brain localization and behavioral impact of the G-protein-gated K+ channel subunit GIRK4.
2000,
Pubmed
Zheng,
Stoichiometry and assembly of olfactory cyclic nucleotide-gated channels.
2004,
Pubmed
,
Xenbase
