Browsing by Subject "G protein"

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    Molecular mechanisms and therapeutic potential of inhibitory G protein signaling in anxiety disorders
    (2020-08) Vo, Baovi
    Anxiety disorders are common and debilitating. Current medications for treating anxiety disorders carry addictive potential and have adverse side effects, highlighting the need for improved therapeutics. Several commonly prescribed drugs used to treat anxiety disorders enhance inhibitory G protein signaling in neurons, leading to the modulation of multiple enzymes and ion channels. The relative contributions of these individual G protein-regulated effectors to anxiety-related behavior are unclear. My thesis research focuses on one such effector – the G protein-gated inwardly rectifying K+ (GIRK) channel. GIRK channels mediate the postsynaptic inhibitory effect of GABA and other inhibitory neurotransmitters in the central nervous system. There are 4 GIRK subunits (GIRK1-4). Neuronal GIRK channels typically contain GIRK1 and GIRK2. Previous work from our lab found that the GIRK channel activator (ML297), which shows a slight preference for GIRK1/2 channels, reduces anxiety-related behavior in mice without exhibiting addictive potential. My central hypothesis is that activators of the GIRK1/2 channel subtype could treat anxiety-related disorders. My thesis explored three interrelated questions: Which brain regions and neuronal populations underlie the influence of GIRK channels on anxiety-related behavior? While ML297 reduces anxiety-related behavior in mice, the relevant brain regions and neuron populations underlying this effect were unclear. I utilized pharmacologic and viral genetic approaches to manipulate GIRK-dependent signaling in distinct neuron populations in the ventral hippocampus (vHPC) and the basolateral amygdala (BLA), and evaluated the impact of these manipulations on anxiety-like behavior using the elevated plus maze (EPM) test. Intra-vHPC ML297 reduced anxiety-related behavior, akin to the effect observed with systemic ML297. In contrast, ML297 infusion into the BLA increased anxiety-related behavior in the EPM. Chemogenetic neuron-specific manipulations revealed neuronal subtypes within vHPC and BLA mediate these effects. These findings could inform targeted treatments for anxiety-related disorders. Do GIRK channel activators have anxiolytic therapeutic potential? Despite the promise of ML297 in studies of anxiety-related behavior, its modest selectivity for neuronal channels, its poor in vivo stability, and its limited ability to penetrate the blood-brain barrier preclude its clinical utility. I characterized a new GIRK channel activator, VU0810464, which showed improved brain penetration and enhanced selectivity for GIRK1/2 channels. I also demonstrated its in vivo efficacy in the stress-induced hyperthermia test. VU0810464 is a new, important tool for investigating the relevance of GIRK1/2 channels in physiology and behavior. What factors influence GIRK-dependent signaling? The GIRK2 subunit is necessary for neuronal GIRK channel function and has three distinct splice variants that have not been extensively characterized. I demonstrated the influence of these splice variants on three different GIRK-dependent signaling pathways in cultured hippocampal neurons using an electrophysiological approach. We found that these GIRK2 splice variants differed in their subcellular distribution, and this difference impacted their contribution to the processing of inhibitory input and to fear learning behavior. This knowledge provides insight into a key element influencing GIRK channel function, and importantly, opens more opportunities for future studies targeting GIRK-dependent signaling for therapeutics. In brief, I present a body of work in this thesis that contributes to the field’s knowledge of GIRK-dependent signaling and offers the potential for novel treatment for anxiety-related disorders.
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    Molecular mechanisms regulating G protein signaling in brain and heart: role of R7 RGS proteins and their binding partners
    (2012-09) Posokhova, Ekaterina N.
    G Protein Coupled Receptor (GPCR) signaling pathways convert signals from the extracellular environment into cellular responses, which is critically important for neurotransmitter action both in central and peripheral nervous systems. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as Regulators of G protein Signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior, and nociception in mammals. One member of R7 RGS family, RGS9-2 has been previously implicated as a key regulator of dopamine and opioid signaling pathways in the basal ganglia of the brain, where it mediates motor control and reward behavior. Dynamic association of RGS9-2 with R7BP (R7 family Binding Protein) is critically important for the regulation of RGS9-2 expression level by proteolytic mechanisms. Changes in RGS9-2 expression are observed in response to a number of signaling events and are thought to contribute to the plasticity of the neurotransmitter action. To unravel the molecular mechanisms regulating levels of RGS9-2 upon its dissociation from R7BP we developed a novel application of the quantitative proteomics approach to monitor interactome dynamics of RGS9-2 in mice. We show that a molecular chaperone HSC70 (Heat Shock Cognate protein 70) identified by this approach is a critical regulator of RGS9-2 expression. HSC70 binds the intrinsically disordered C-terminal domain of RGS9-2 upon the dissociation of R7BP/RGS9-2 complex, and targets the complex to degradation. In addition to their critical role in shaping neurotransmitter response in the brain, RGS proteins can regulate function of peripheral organs by modulating their responses to the influences of autonomic nervous system. The role of RGS proteins in the regulation of cardiac function and heart rate has received significant attention in the recent years. With over 30 RGS proteins identified, their specific roles in heart physiology remain to be established. Parasympathetic autonomic influence plays an important role in shaping cardiac output acting to decrease heart rate and counteract the pro-arrhythmic effects of sympathetic activation. Acetylcholine (ACh) released from post-ganglionic parasympathetic neurons activates M2 muscarinic receptor (M2R) and its downstream effector, potassium channel IKACh, in pacemaker cells and atrial myocytes. This leads to cell hyperpolarization and ultimately, decreased heart rate (HR). The second part of the dissertation demonstrates cardiac expression of RGS6 member of R7 RGS family, which has been previously thought to be a neuron-specific regulator. Elimination of RGS6 in mice results in potentiated M2R-IKACh signaling, as evidenced by prolonged deactivation kinetics of IKACh in cardiomyocytes, mild resting bradycardia, and augmented HR deceleration in response to M2R activation. Furthermore, RGS6 specifically co-precipitates with one of the two subunits of IKACh, GIRK4 in transfected HEK293 cells. Direct binding to the effector channel might serve to facilitate RGS6-mediated modulation of parasympathetic influence on atrial myocytes and in mice. Altogether, the findings comprising this dissertation demonstrate a novel role of RGS6 in regulation of cardiac function, as well as two novel protein-protein interactions of R7 RGS proteins. Identified protein complexes influence G protein signaling by either (i) altering the availability of the regulator (RGS9-2/HSC70), or (ii) by serving to co-localize the major pathway components (RGS6/GIRK4).