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 ␤␥ subunit (G␤␥) 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 GIRK1͞GIRK4 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 GIRK1͞GIRK4 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.play an important role in controlling membrane excitability and synaptic transmission (1, 2). Four members of GIRK channels have been cloned in mammals, i.e., GIRK1 through GIRK4 (Kir3.1 through Kir3.4). These channels are expressed in the heart, brain, and endocrine tissues (3, 4). Stoichiometric studies indicate that a functional GIRK channel consists of four homomeric subunits or two pairs of heteromeric subunits (3, 4). Coassembly of GIRK1 with GIRK4 forms muscarinic receptorcoupled K ϩ channels mainly in the heart (2, 5). The GIRK channels are activated by certain inhibitory transmitters and hormones. On activation of G i/o -coupled receptors by the neurotransmitters or hormones, the G protein ␤␥ subunit (G ␤␥ ) dissociated from the heterotrimeric G ␣␤␥ directly interacts with cytosolic domains of GIRK channel proteins (6-9), and activates the channels through a mechanism that involves movement of pore-lining helices (10-12). Critical domains and amino acids have been identified to be responsible for the basal G ␤␥ -dependent and agonist-induced channel activities (6,7,13,14). Furthermore, the GIRK channel activation may rely on local phosphatidylinositol-4,5-biphosphate (PIP 2 ) in membrane microdomains (15, 16) or cytosolic Na ϩ (16,17).In addition to direct activation by G ␤␥ , GIRK channels are inhibited by a number of excitatory neurotransmitters or hormones, such as acetylcholine (18-20), thyroid-stimulating hormone (TSH)-releasing hormones (21), bombesin (22), substance P (SP) (23), and glutamate (24). These transmitters or hormones sh...

Reduced 5-HT1A- and GABABReceptor Function in Dorsal Raphé Neurons Upon Chronic Fluoxetine Treatment of Socially Stressed Rats

“…Alternative mechanisms could be through PKC-dependent phosphorylation of GIRK channels (Mao et al 2004), depletion of PIP2, an endogenous co-factor necessary for GIRK channel activity (Cho et al 2005;Lei et al 2003) or through a glucocorticoid-mediated modulation of GIRK responsiveness (Fairchild et al 2003). Recently the background potassium channel TREK-1 was proposed as a target for antidepressant action in DRN neurons (Heurteaux et al 2006).…”

Dopamine D2 Receptor Modulation of K+ Channel Activity Regulates Excitability of Nucleus Accumbens Neurons at Different Membrane Potentials

“…Second, PIP 2 is hydrolyzed by PLC to form IP 3 and diacylglycerol (DAG). DAG activates protein kinase C (PKC), which can also inhibit K ir channel activity (Mao et al 2004;Stevens et al 1999) that should also decrease the inward rectification. Given the above, we propose that the D 2 Rmediated reduction in the inward rectification (representing decreased I Kir ) should be attributed to activation of PI-PLC and a consequent reduction of intracellular PIP 2 levels.…”

Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

“…Oocytes were obtained from the frog X. laevis (Mao et al, 2004;Xu et al, 2001). Frogs were anesthetized by bathing in 0.3% 3-aminobenzoic acid ethyl ester.…”