title: Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq.
abstract: Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor-mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of Galphaq and Galpha11, but not Galpha13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPbetaS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPbetaS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPbetaS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein-stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, G-424 Health Sciences Building, Box 357290, Seattle, WA 98195-7290, USA.
authors.forename: Byung-Chang
authors.initials: BC
authors.lastname: Suh
authors.forename: Lisa F
authors.initials: LF
authors.lastname: Horowitz
authors.forename: Wiebke
authors.initials: W
authors.lastname: Hirdes
authors.forename: Ken
authors.initials: K
authors.lastname: Mackie
authors.forename: Bertil
authors.initials: B
authors.lastname: Hille
mesh_terms: D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D003001:Cloning, Molecular; D003198:Computer Simulation; D004553:Electric Conductivity; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D007668:Kidney / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D024642:Potassium Channels, Voltage-Gated / Q000187:drug effects / Q000502:physiology*; D043643:Receptor, Muscarinic M1 / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013329:Structure-Activity Relationship
chemical_list: D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; D051659:KCNQ3 Potassium Channel; C496916:KCNQ3 protein, human; D024642:Potassium Channels, Voltage-Gated; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D008274:Magnesium