title: Mechanosensitivity of GIRK channels is mediated by protein kinase C-dependent channel-phosphatidylinositol 4,5-bisphosphate interaction.
abstract: Gprotein-activated inwardly rectifying K+ channel (GIRK or Kir3) currents are inhibited by mechanical stretch of the cell membrane, but the underlying mechanisms are not understood. In Xenopus oocytes heterologously expressing GIRK channels, membrane stretch induced by 50% reduction of osmotic pressure caused a prompt reduction of GIRK1/4, GIRK1, and GIRK4 currents by 16.6-42.6%. Comparable GIRK current reduction was produced by protein kinase C (PKC) activation (phorbol 12-myristate 13-acetate). The mechanosensitivity of the GIRK4 current was abolished by pretreatment with PKC inhibitors (staurosporine or calphostin C). Neither hypo-osmotic challenge nor PKC activation affected IRK1 currents. GIRK4 chimera (GIRK4-IRK1-(Lys207-Leu245)) and single point mutant (GIRK4(I229L)), in which the phosphatidylinositol 4,5-bisphosphate (PIP2) binding domain or residue was replaced by the corresponding region of IRK1 to strengthen the channel-PIP2 interaction, showed no mechanosensitivity and minimal PKC sensitivity. IRK1 gained mechanosensitivity and PKC sensitivity by reverse double point mutation of the PIP2 binding domain (L222I/R213Q). Overexpression of Gbetagamma, which is known to strengthen the channel-PIP2 interaction, attenuated the mechanosensitivity of GIRK4 channels. In oocytes expressing a pleckstrin homology domain of PLC-delta tagged with green fluorescent protein, hypo-osmotic challenge or PKC activation caused a translocation of the fluorescence signal from the cell membrane to the cytosol, reflecting PIP2 hydrolysis. The translocation was prevented by pretreatment with PKC inhibitors. Involvement of PKC activation in the mechanosensitivity of muscarinic K+ channels was confirmed in native rabbit atrial myocytes. These results suggest that the mechanosensitivity of GIRK channels is mediated primarily by channel-PIP2 interaction, with PKC playing an important role in modulating the interaction probably through PIP2 hydrolysis.
authors.affiliation: Department of Circulation, Research Institute of Environmental Medicine, Nagoya University, Nagoya, 464-8601, Japan.
authors.forename: Liyan
authors.initials: L
authors.lastname: Zhang
authors.forename: Jong-Kook
authors.initials: JK
authors.lastname: Lee
authors.forename: Scott A
authors.initials: SA
authors.lastname: John
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Uozumi
authors.forename: Itsuo
authors.initials: I
authors.lastname: Kodama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D049452:Green Fluorescent Proteins; D006868:Hydrolysis; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009281:Naphthalenes / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D009995:Osmosis; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D017354:Point Mutation; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014981:Xenopus; D014982:Xenopus laevis
chemical_list: D004791:Enzyme Inhibitors; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D008164:Luminescent Proteins; D009281:Naphthalenes; D019269:Phosphatidylinositol 4,5-Diphosphate; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D018075:RNA, Complementary; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate