Gbetagamma-dependent and Gbetagamma-independent basal activity of G protein-activated K+ channels.
Cardiac and neuronal G protein-activated K+ channels (GIRK; Kir3) open following the binding of Gbetagamma subunits, released from Gi/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free Gbetagamma, but a Gbetagamma-independent component has also been envisaged. We investigated Gbetagamma dependence of the basal GIRK activity (A(GIRK,basal)) quantitatively, by titrated expression of Gbetagamma scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used Gbetagamma scavenger, myristoylated C terminus of beta-adrenergic kinase (m-cbetaARK), reduced A(GIRK,basal) by 70-80% and eliminated the acetylcholine-evoked current (I(ACh)). However, we found that m-cbetaARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed Gbetagamma scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents. m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of A(GIRK,basal) by up to 90%. Expression of GIRK was accompanied by an increase in the level of Gbetagamma and Galpha in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of Gbetagamma and its constitutive association with GIRK may underlie the excessively high A(GIRK,basal) observed at high expression levels of GIRK. Only 10-15% of A(GIRK,basal) persisted upon expression of both m-phosducin and cbetaARK. These results demonstrate that a major part of Ibasal is Gbetagamma-dependent at all levels of channel expression, and only a small fraction (<10%) may be Gbetagamma-independent.
Rishal
Ida
I
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel.
Porozov
Yuri
Y
Yakubovich
Daniel
D
Varon
Dalia
D
Dascal
Nathan
N
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005136:Eye Proteins / Q000737:chemistry; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000502:physiology*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D019814:Myristic Acid / Q000737:chemistry / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D051381:Rats; D013997:Time Factors; D014981:Xenopus
D018076:DNA, Complementary; D005136:Eye Proteins; C118981:G-protein Beta gamma; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D010750:Phosphoproteins; D015221:Potassium Channels; C065631:phosducin; D019814:Myristic Acid; D012313:RNA; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D011188:Potassium