Project

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Regulation of GABA(B) receptor cell surface expression by endocytosis under normal and pathophysiological conditions

English title Regulation of GABA(B) receptor cell surface expression by endocytosis under normal and pathophysiological conditions
Applicant Benke Dietmar
Number 121963
Funding scheme Project funding
Research institution Institut für Pharmakologie und Toxikologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Neurophysiology and Brain Research
Start/End 01.11.2008 - 31.10.2011
Approved amount 288'000.00
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All Disciplines (3)

Discipline
Neurophysiology and Brain Research
Cellular Biology, Cytology
Pharmacology, Pharmacy

Keywords (10)

inhibitory neurotransmission; GABA(B) receptor; endocytosis; receptor associated proteins; recycling; degradation; trafficking; exocytosis, sorting; neuron; neuropathic pain

Lay Summary (English)

Lead
Lay summary
GABA is the main inhibitory neurotransmitter responsible for balancing excitatory neurotransmission in the mammalian central nervous system. GABA conveys slow inhibitory transmission via the G protein-coupled GABA(B) receptors. GABA(B) receptors are abundantly expressed in the brain and have been implicated as potential targets for the treatment of a variety of neurological disorders such as epilepsy, spasticity, addiction, schizophrenia, depression, anxiety and chronic pain. Elucidation of the regulation of GABA(B) receptor signaling is essential for understanding the contribution of GABA(B) receptors to pathophysiological conditions such as chronic pain. In particular, regulation of the number of GABA(B) receptors at the cell surface is expected to be a main factor that influences their signaling strength. However, the involved processes are as yet largely unknown. In this research project, we put forward the hypothesis that regulated endocytosis is a powerful mechanism to adjust GABA(B) receptor cell surface expression to changing physiological conditions. It is our aim to gain insights into the mechanisms of endocytosis, intracellular sorting and degradation of GABA(B) receptors under normal and pathophysiological conditions. To this end we have shown that GABA(B) receptors constitutively internalize via clathrin-dependent endocytosis and are sorted to two distinct and independent intracellular routes. Internalized GABA(B) receptors are either targeted to lysosomes for degradation or are recycled back to the plasma membrane. Our results indicate that recycling of GABA(B) receptors represent a highly regulated mechanism that enables neurons to rapidly adjust the density of cell surface GABA(B) receptors in response to external or internal stimuli. Using a combination of biochemical, molecular cell biology and immunocytochemical imaging techniques we aim at further elucidating the mechanisms that regulates cell surface numbers of GABA(B) receptors in neurons under physiological and the pathophysiological state of neuropathic pain. The results of these studies are expected to significantly advance our understanding of the regulation of GABA(B) receptors. They may lead to a better understanding of the involvement of GABA(B) receptors in chronic pain and, depending on the outcome, may point at novel therapeutic strategies for its treatment.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Mechanisms of GABAB receptor exocytosis, endocytosis, and degradation.
Benke Dietmar (2010), Mechanisms of GABAB receptor exocytosis, endocytosis, and degradation., in Advances in pharmacology (San Diego, Calif.), 58, 93-111.
Sustained glutamate activation downregulates GABAB receptors by shifting the balance from recycling to lysosomal degradation
Maier Patrick J., Marin Isabel, Grampp Thamas, Sommer Andrea, Benke Dietmar (2010), Sustained glutamate activation downregulates GABAB receptors by shifting the balance from recycling to lysosomal degradation, in Jounal of Biological Chemistry, 258(46), 35606-35614.
Modulation of cell surface GABAB receptors by desensitization, trafficking and regulated degradation
Benke Dietmar, Zemoura Khaled, Maier Patrick J., Modulation of cell surface GABAB receptors by desensitization, trafficking and regulated degradation, in World Journal of Biological Chemistry.

Associated projects

Number Title Start Funding scheme
138382 Regulation of cell surface GABA(B) receptors by trafficking mechanisms: contribution to synaptic plasticity and disease states 01.11.2011 Project funding

Abstract

GABA(B) receptors are G protein-coupled receptors that mediate slow inhibitory neurotransmission by controlling postsynaptically the membrane potential via activation of K+ channels and presynaptically the transmitter release via inhibition of Ca2+ channels. GABA(B) receptors are abundantly expressed in the nervous system and have been implicated as potential targets for the treatment of several neurological disorders such as epilepsy, spasticity, addiction, schizophrenia, depression, anxiety and chronic pain. Elucidation of the regulation of GABA(B) receptor signaling is essential for understanding the contribution of GABA(B) receptors to pathophysiological conditions such as chronic pain. In particular, regulation of the number of GABA(B) receptors at the cell surface is expected to be a main factor that influences their signaling strength. However, the involved processes are as yet largely unknown. In this proposal, we put forward the hypothesis that regulated endocytosis is a powerful mechanism to adjust GABA(B) receptor cell surface expression to changing physiological conditions. It is our aim to gain insights into the mechanisms of endocytosis, intracellular sorting and degradation of GABA(B) receptors under normal and pathophysiological conditions. The proposal is divided into five interrelated subprojects:1. The mechanisms that regulate GABA(B) receptor endocytosis and sorting will be analyzed in cultured cortical neurons. We will focus on the hypothesis that neuronal activity and activity-dependent processes such as phosphorylation and ubiquitination modulate endocytosis, sorting and/or degradation of GABA(B) receptors. In particular, regulating recycling of receptors may provide a powerful mechanism to instantly adjust cell surface expression of receptors to changing physiological conditions. 2. Endocytosis and sorting of receptors require a complex sequence of protein-protein interactions. In the second subproject, we aim at identifying GABA(B) receptor interacting proteins important for endocytosis and sorting. In a first approach, we will analyze by coimmunoprecipitation the association of GABA(B) receptors with proteins already known to be involved in these processes from other receptor systems. A second line of experiments aims at the identification of novel GABA(B) receptor interacting proteins by using pull-down experiments and subsequent identification of proteins by mass spectroscopy. 3. Preliminary data indicate the involvement of the ubiquitin-proteasome system in sorting and degradation of GABA(B) receptors. In the third project we will analyze the contribution of the ubiquitin-proteasome system to these processes using biochemical and immunofluorescence studies on cultured neurons. 4. The analysis of receptor endocytosis is predominantly done on cultured primary cells or cell lines. However, in the case of neurons these in vitro systems lack at least natural connectivity and network properties. Therefore, in the fourth subproject, we aim at analyzing GABA(B) receptor endocytosis in a more native condition, the acute brain slice preparation. This method will enable us to analyze endocytosis of GABA(B) receptors in different brain areas and after electrical stimulation of defined pathways.5. In the fifth project, we aim at analyzing potential alternations of GABA(B) receptor endocytosis under pathophysiological conditions. GABA(B) receptors are known to regulate nociception but direct evidence of its involvement in the development and/or maintenance in chronic pain is lacking. Using the acute spinal cord slice preparation and an animal model of neuropathic pain we will analyze whether alterations in endocytotic parameters may be a factor contributing to chronic pain. The results of these studies are expected to significantly advance our understanding of the regulation of GABA(B) receptors. They may lead to a better understanding of the involvement of GABA(B) receptors in chronic pain and, depending on the outcome, may point at novel therapeutic strategies for its treatment.
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