Dataset Information

Morphine- and CaMKII-dependent enhancement of GIRK channel signaling in hippocampal neurons.

ABSTRACT: G-protein-gated inwardly rectifying potassium (GIRK) channels, which help control neuronal excitability, are important for the response to drugs of abuse. Here, we describe a novel pathway for morphine-dependent enhancement of GIRK channel signaling in hippocampal neurons. Morphine treatment for ?20 h increased the colocalization of GIRK2 with PSD95, a dendritic spine marker. Western blot analysis and quantitative immunoelectron microscopy revealed an increase in GIRK2 protein and targeting to dendritic spines. In vivo administration of morphine also produced an upregulation of GIRK2 protein in the hippocampus. The mechanism engaged by morphine required elevated intracellular Ca(2+) and was insensitive to pertussis toxin, implicating opioid receptors that may couple to Gq G-proteins. Met-enkephalin, but not the ?-selective (DAMGO) and ?-selective (DPDPE) opioid receptor agonists, mimicked the effect of morphine, suggesting involvement of a heterodimeric opioid receptor complex. Peptide (KN-93) inhibition of CaMKII prevented the morphine-dependent change in GIRK localization, whereas expression of a constitutively activated form of CaMKII mimicked the effects of morphine. Coincident with an increase in GIRK2 surface expression, functional analyses revealed that morphine treatment increased the size of serotonin-activated GIRK currents and Ba(2+)-sensitive basal K(+) currents in neurons. These results demonstrate plasticity in neuronal GIRK signaling that may contribute to the abusive effects of morphine.

SUBMITTER: Nassirpour R

PROVIDER: S-EPMC3010279 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Morphine- and CaMKII-dependent enhancement of GIRK channel signaling in hippocampal neurons.

Nassirpour Rounak R Bahima Laia L Lalive Arnaud L AL Lüscher Christian C Luján Rafael R Slesinger Paul A PA

The Journal of neuroscience : the official journal of the Society for Neuroscience 20101001 40

G-protein-gated inwardly rectifying potassium (GIRK) channels, which help control neuronal excitability, are important for the response to drugs of abuse. Here, we describe a novel pathway for morphine-dependent enhancement of GIRK channel signaling in hippocampal neurons. Morphine treatment for ∼20 h increased the colocalization of GIRK2 with PSD95, a dendritic spine marker. Western blot analysis and quantitative immunoelectron microscopy revealed an increase in GIRK2 protein and targeting to d ...[more]

PMID: 20926668

Similar Datasets

Project description:Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K+ or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within ~ 30-40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core, ~ 100-120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~ 60-70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60-75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68-77 nm), than in control (59-63 nm) and stimulated samples (49-53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

Project description:BackgroundThe large conductance Ca(2+) - and voltage-activated K(+) channel (BK) is an important player in molecular and behavioral alcohol tolerance. Trafficking and surface expression of ion channels contribute to the development of addictive behaviors. We have previously reported that internalization of the BK channel is a component of molecular tolerance to ethanol (EtOH).MethodsUsing primary cultures of hippocampal neurons, we combine total internal reflection fluorescence microscopy, electrophysiology, and biochemical techniques to explore how exposure to EtOH affects the expression and subcellular localization of endogenous BK channels over time.ResultsExposure to EtOH changed the expression of endogenous BK channels in a time-dependent manner at the perimembrane area (plasma membrane and/or the area adjacent to it), while total protein levels of BK remain unchanged. These results suggest a redistribution of the channel within the neurons rather than changes in synthesis or degradation rates. Our results showed a temporally nonlinear effect of EtOH on perimembrane expression of BK. First, there was an increase in BK perimembrane expression after 10 minutes of EtOH exposure that remained evident after 3 hours, although not correlated to increases in functional channel expression. In contrast, after 6 hours of EtOH exposure, we observed a significant decrease in both BK perimembrane expression and functional channel expression. Furthermore, after 24 hours of EtOH exposure, perimembrane levels of BK had returned to baseline.ConclusionsWe report a complex time-dependent pattern in the effect of EtOH on BK channel trafficking, including successive increases and decreases in perimembrane expression and a reduction in active BK channels after 3 and 6 hours of EtOH exposure. Possible mechanisms underlying this multiphasic trafficking are discussed. As molecular tolerance necessarily underlies behavioral tolerance, the time-dependent alterations we see at the level of the channel may be relevant to the influence of drinking patterns on the development of behavioral tolerance.

Project description:In the hippocampus, signaling through G protein-coupled receptors is modulated by Regulators of G protein signaling (Rgs) proteins, which act to stimulate the rate of GTP hydrolysis, and consequently, G protein inactivation. The R7-Rgs subfamily selectively deactivates the G(i/o)-class of Gα subunits that mediate the action of several GPCRs. Here, we used co-immunoprecipitation, electrophysiology and immunoelectron microscopy techniques to investigate the formation of macromolecular complexes and spatial relationship of Rgs7/Gβ5 complexes and its prototypical signaling partners, the GABAB receptor and Girk channel. Co-expression of recombinant GABAB receptors and Girk channels in combination with co-immunoprecipitation experiments established that the Rgs7/Gβ5 forms complexes with GABAB receptors or Girk channels. Using electrophysiological experiments, we found that GABAB -Girk current deactivation kinetics was markedly faster in cells coexpressing Rgs7/Gβ5. At the electron microscopic level, immunolabeling for Rgs7 and Gβ5 proteins was found primarily in the dendritic layers of the hippocampus and showed similar distribution patterns. Immunoreactivity was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of pyramidal cells and, to a lesser extent, to that of presynaptic terminals. Quantitative analysis of immunogold particles for Rgs7 and Gβ5 revealed an enrichment of the two proteins around excitatory synapses on dendritic spines, virtually identical to that of Girk2 and GABAB1 . These data support the existence of macromolecular complexes composed of GABAB receptor-G protein-Rgs7-Girk channels in which Rgs7 and Gβ5 proteins may preferentialy modulate GABAB receptor signaling through the deactivation of Girk channels on dendritic spines. In contrast, Rgs7 and Girk2 were associated but mainly segregated from GABAB1 in dendritic shafts, where Rgs7/Gβ5 signaling complexes might modulate Girk-dependent signaling via a different metabotropic receptor(s).

Dataset Information

Morphine- and CaMKII-dependent enhancement of GIRK channel signaling in hippocampal neurons.

Publications

Morphine- and CaMKII-dependent enhancement of GIRK channel signaling in hippocampal neurons.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets