Gbetagamma-dependent and Gbetagamma-independent basal activity of G protein-activated K+ channels

J Biol Chem. 2005 Apr 29;280(17):16685-94. doi: 10.1074/jbc.M412196200. Epub 2005 Feb 23.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylcholine / pharmacology
  • Animals
  • Blotting, Western
  • Cattle
  • Cell Membrane / metabolism
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Eye Proteins / chemistry
  • GTP-Binding Protein Regulators
  • GTP-Binding Protein beta Subunits / chemistry
  • GTP-Binding Protein beta Subunits / physiology*
  • GTP-Binding Protein gamma Subunits / chemistry
  • GTP-Binding Protein gamma Subunits / physiology*
  • GTP-Binding Proteins / chemistry*
  • Genetic Vectors
  • Glutathione Transferase / metabolism
  • Humans
  • Mice
  • Microscopy, Confocal
  • Myristic Acid / chemistry
  • Myristic Acid / metabolism
  • Oocytes / metabolism
  • Phosphoproteins / chemistry
  • Potassium / chemistry
  • Potassium Channels / chemistry*
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA / chemistry
  • Rats
  • Time Factors
  • Xenopus

Substances

  • DNA, Complementary
  • Eye Proteins
  • G-protein Beta gamma
  • GTP-Binding Protein Regulators
  • GTP-Binding Protein beta Subunits
  • GTP-Binding Protein gamma Subunits
  • Phosphoproteins
  • Potassium Channels
  • phosducin
  • Myristic Acid
  • RNA
  • Glutathione Transferase
  • GTP-Binding Proteins
  • Acetylcholine
  • Potassium