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Ongoing Research Support - Chavkin (PI): Models of Opioid Receptor Desensitization Mechanisms R37 DA11672-09 The primary goal
of this project is to define the specific sites of mu, delta and kappa opioid
receptor phosphorylation responsible for kinase-induced receptor
desensitization. The effects on receptor function of the different G protein
coupled receptor kinases (GRKs) will be compared to other, nonspecific
kinases (e.g. PKA, CamKII, PKC) in opioid receptor desensitization processes.
Agonist-dependent mechanisms regulating opioid responses in three models of
neuronal function: receptor-transfected cells, Xenopus oocytes expressing opioid receptors, and
opioid-responsive neurons in mammalian brain slices. Results obtained will be
compared to define the processes underlying homologous opioid receptor
desensitization. Molecular Components Underlying Drug Abuse P01 DA15916-05 This is a
NIDA-Program project grant supporting four projects (Chavkin, Mackie,
Palmiter and McKnight), four Cores (Administrative, Mouse Genetics,
Behavioral, Anatomical) and a pilot research project program. The program
supports the generation and characterization (e.g. behavioral, physiological,
and anatomical) of mice having tissue-specific, inducible modifications
(mutations or deletions) of genes thought to be important for opioid,
cannabinoid or psychostimulant drug action. Chavkin's project is titled
"Components underlying opioid receptor tolerance." This project
specifically addresses sites within the mu opioid receptor important for
morphine tolerance. Opioid Mediation of Stress-Potentiated Cocaine Response R01 DA016898-05 The primary goal
of this project is to identify the role of endogenous kappa opioid systems in
mediating the potentiating effects of chronic behavioral stress on the
rewarding properties of cocaine. The work involves behavioral assays
(stress-induced analgesia & immobility, conditioned place preference, and
drug self-administration) using transgenic mice. Additional immunohistochemical
studies will define the anatomical locatization of kappa opioid receptor
activated by behavioral stress, and subsequent electrophysiological studies
will define the effects of stress on kappa opioid responses in the responsive
brain regions (e.g. VTA and NAc). Training in Molecular Pharmacology of Abused Drugs T32 DA07278-14 This
institutional training grant supports 2 postdoctoral fellows and 4
predoctoral fellows per year. General Description: The research effort in the lab continues to be focused on 1) the
mechanisms regulating synaptic transmission in the mammalian brain and 2) the
molecular mechanisms regulating opioid receptor functioning. We use a
combination of electrophysiological, anatomical, and molecular approaches to
the understanding of the role of opioid neuropeptides as neurotransmitters in
the hippocampus. We know that these endogenous peptides regulate synaptic
plasticity in memory events, and we know that changes in the physiological
role of the endogenous opioids occurs in certain forms of epilepsy. Our work
has attempted to rigorously define the anatomical and biophysical properties
of this neuropeptide synapse in the mammalian brain. We express opioid receptor clones along with potassium channels in Xenopus oocytes and mammalian cell lines to study the
signal transduction events responsible for opiate drug actions. Coupling
between the receptor and the channel is not static and can be regulated by
specific kinases and accessory proteins. This regulation is likely to be important
to opiate tolerance and sensitivity to both pain and stress. Phosphorylation
of specific serine, threonine and tyrosine residues within the opioid
receptor changes receptor functioning in vitro, and we are presently exploring how those changes
affect the animal's responses to drugs, pain, and stress using transgenic
mice. To understand the biochemical coupling between opioid receptors and ion
channels important for opioid actions, we have study the cellular processes
mediating opioid effects in hippocampal slices and cultures. Opioid receptor
activation leads to the activation of pertussis toxin sensitive G proteins
that then cause an increase in potassium channel conductance and decrease in
calcium channel conductance. We are defining the types of potassium channels
activated. Changes in the activation of specific ion channels results in a
somatic and presynaptic inhibition by molecular mechanisms that we are
defining. To understand how the biochemical coupling between receptor and ion
channel is regulated, we are measuring the effects of prolonged activation of
opioid receptors. The desensitization of the response following extended
agonist exposure is a complex mixture of events including changes receptor
phosphorylation, changes in G-protein levels, and changes in the channel. The
important steps in this regulation are being identified and defined. We have
continued to use the oocyte gene expression system and transfected
neuroblastoma cell lines to reconstitute the opioid receptor link to potassium
channels as defined in the hippocampus and to study the actions of potential
regulators by co-expression. The results of these studies are expected to
provide the necessary understanding required to define animal behavior at the
cellular and molecular level. The goal of this project is to define the structural features of the
opioid receptors responsible for the homologous desensitization mediated by G
protein coupled receptor kinases and arrestins. Site-directed mutagenesis of
receptors and coexpression of the signal transduction components (e.g.
receptors, kinases, arrestins, and channels) in Xenopus oocytes will be done.
The second half of the proposed studies involves whole cell voltage clamp
recording of identified opioid-sensitive interneurons in the hippocampal
slice to define the kinetics of homologous desensitization in real cells. |