title: Molecular basis for the inhibition of G protein-coupled inward rectifier K(+) channels by protein kinase C.
abstract: G protein-coupled inward rectifier K(+) (GIRK) channels regulate cellular excitability and neurotransmission. The GIRK channels are activated by a number of inhibitory neurotransmitters through the G protein betagamma subunit (G(betagamma)) after activation of G protein-coupled receptors and inhibited by several excitatory neurotransmitters through activation of phospholipase C. If the inhibition is produced by PKC, there should be PKC phosphorylation sites in GIRK channel proteins. To identify the PKC phosphorylation sites, we performed systematic mutagenesis analysis on GIRK4 and GIRK1 subunits expressed in Xenopus oocytes. Our data showed that the heteromeric GIRK1/GIRK4 channels were inhibited by a PKC activator phorbol 12-myristate 13-acetate (PMA) through reduction of single channel open-state probability. Direct application of the catalytic subunit of PKC to excised patches had a similar inhibitory effect. This inhibition was greatly eliminated by mutation of Ser-185 in GIRK1 and Ser-191 in GIRK4 that remained G protein sensitive. The PKC-dependent phosphorylation seems to mediate the channel inhibition by the excitatory neurotransmitter substance P (SP) as specific PKC inhibitors and mutation of these PKC phosphorylation sites abolished the SP-induced inhibition of GIRK1/GIRK4 channels. Thus, these results indicate that the PKC-dependent phosphorylation underscores the inhibition of GIRK channels by SP, and Ser-185 in GIRK1 and Ser-191 in GIRK4 are the PKC phosphorylation sites.
authors.affiliation: Department of Biology, Georgia State University, 24 Peachtree Center Avenue, Atlanta, GA 30302-4010, USA.
authors.forename: Jinzhe
authors.initials: J
authors.lastname: Mao
authors.forename: Xueren
authors.initials: X
authors.lastname: Wang
authors.forename: Fuxue
authors.initials: F
authors.lastname: Chen
authors.forename: Runping
authors.initials: R
authors.lastname: Wang
authors.forename: Asheebo
authors.initials: A
authors.lastname: Rojas
authors.forename: Yun
authors.initials: Y
authors.lastname: Shi
authors.forename: Hailan
authors.initials: H
authors.lastname: Piao
authors.forename: Chun
authors.initials: C
authors.lastname: Jiang
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013373:Substance P / Q000502:physiology
chemical_list: D024661:Potassium Channels, Inwardly Rectifying; D013373:Substance P; D011493:Protein Kinase C; D019204:GTP-Binding Proteins