Recruitment of Gβγ controls the basal activity of G-protein coupled inwardly rectifying potassium (GIRK) channels: crucial role of distal C terminus of GIRK1.
The G-protein coupled inwardly rectifying potassium (GIRK, or Kir3) channels are important mediators of inhibitory neurotransmission via activation of G-protein coupled receptors (GPCRs). GIRK channels are tetramers comprising combinations of subunits (GIRK1-4), activated by direct binding of the Gβγ subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, Gβγ-dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed Gβγ. Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by Gβγ. The high Ibasal of GIRK1 seems to be associated with its unique distal C terminus (G1-dCT), which is not present in the other subunits. We investigated the role of G1-dCT using electrophysiological and fluorescence assays in Xenopus laevis oocytes and protein interaction assays. We show that expression of GIRK1/2 increases the plasma membrane level of coexpressed Gβγ (a phenomenon we term 'Gβγ recruitment') but not of coexpressed Gαi3. All GIRK1-containing channels, but not GIRK2 homomers, recruited Gβγ to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and Gβγ, but not Gαi3. Nevertheless, the truncation of G1-dCT does not impair activation by Gβγ. In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes Gβγ recruitment and decreases Ibasal. Thus, we conclude that G1-dCT carries an essential role in Gβγ recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a Gβγ anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by Gβγ.
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
Uri
U
Kahanovitch
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
Vladimir
V
Tsemakhovich
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
Shai
S
Berlin
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
Moran
M
Rubinstein
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
Boaz
B
Styr
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
Ruth
R
Castel
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
Sagit
S
Peleg
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
Galit
G
Tabak
Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX, 77030, USA.
Carmen W
CW
Dessauer
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel.
Tatiana
T
Ivanina
Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel dascaln@post.tau.ac.il uri.kahanovitch@gmail.com.
Nathan
N
Dascal
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D015640:Ion Channel Gating; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D014981:Xenopus
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D021122:Protein Subunits