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Ongoing Research Support - Chavkin (PI): Models of Opioid Receptor Desensitization Mechanisms R37 DA11672-10 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. 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 DA07278-15 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. |