Regulator of G Protein Signaling Protein Suppression of G o Protein-Mediated 2A Adrenergic Receptor Inhibition of Mouse Hippocampal CA3 Epileptiform Activity

Department of Pharmacology, Physiology and Therapeutics, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58202-9037, USA.
Molecular pharmacology (Impact Factor: 4.13). 02/2009; 75(5):1222-30. DOI: 10.1124/mol.108.054296
Source: PubMed


Activation of G protein-coupled alpha(2) adrenergic receptors (ARs) inhibits epileptiform activity in the hippocampal CA3 region. The specific mechanism underlying this action is unclear. This study investigated which subtype(s) of alpha(2)ARs and G proteins (Galpha(o) or Galpha(i)) are involved in this response using recordings of mouse hippocampal CA3 epileptiform bursts. Application of epinephrine (EPI) or norepinephrine (NE) reduced the frequency of bursts in a concentration-dependent manner: (-)EPI > (-)NE > (+)NE. To identify the alpha(2)AR subtype involved, equilibrium dissociation constants (pK(b)) were determined for the selective alphaAR antagonists atipamezole (8.79), rauwolscine (7.75), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB-4101; 6.87), and prazosin (5.71). Calculated pK(b) values correlated best with affinities determined previously for the mouse alpha(2A)AR subtype (r = 0.98, slope = 1.07). Furthermore, the inhibitory effects of EPI were lost in hippocampal slices from alpha(2A)AR-but not alpha(2C)AR-knockout mice. Pretreatment with pertussis toxin also reduced the EPI-mediated inhibition of epileptiform bursts. Finally, using knock-in mice with point mutations that disrupt regulator of G protein signaling (RGS) binding to Galpha subunits to enhance signaling by that G protein, the EPI-mediated inhibition of bursts was significantly more potent in slices from RGS-insensitive Galpha(o)(G184S) heterozygous (Galpha(o)+/GS) mice compared with either Galpha(i2)(G184S) heterozygous (Galpha(i2)+/GS) or control mice (EC(50) = 2.5 versus 19 and 23 nM, respectively). Together, these findings indicate that the inhibitory effect of EPI on hippocampal CA3 epileptiform activity uses an alpha(2A)AR/Galpha(o) protein-mediated pathway under strong inhibitory control by RGS proteins. This suggests a possible role for RGS inhibitors or selective alpha(2A)AR agonists as a novel antiepileptic drug therapy.

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    • "If enhanced Ga o -mediated presynaptic inhibition occurred in inhibitory (GABAergic) neurons, as has been proposed for the epileptogenic Nav1.1 LOF mutants (Yu et al. 2006), one could see hyper-excitability due to suppressed inhibitory tone. This is consistent with our prior observation of reduced in vitro excitability in hippocampal slices from mice carrying our mutation (Goldenstein et al. 2009). In that model, the ability of epinephrine to suppress hippocampal epileptiform discharges was enhanced in the Ga o "
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    ABSTRACT: Recent evidence suggests that G protein-coupled receptors, especially those linked to G(alpha)(i), contribute to the mechanisms of anesthetic action. Regulator of G protein signaling (RGS) proteins bind to activated G(alpha)(i) and inhibit signal transduction. Genomic knock-in mice with an RGS-insensitive G(alpha)(i2) G184S (G(alpha)(i2) GS) allele exhibit enhanced G(alpha)(i2) signaling and provide a novel approach for investigating the role of G(alpha)(i2) signaling and RGS proteins in general anesthesia. We anesthetized homozygous G(alpha)(i2) GS/GS and wild-type (WT) mice with isoflurane and quantified time (in seconds) to loss and resumption of righting response. During recovery from isoflurane anesthesia, breathing was quantified in a plethysmography chamber for both lines of mice. G(alpha)(i2) GS/GS mice required significantly less time for loss of righting and significantly more time for resumption of righting than WT mice. During recovery from isoflurane anesthesia, G(alpha)(i2) GS/GS mice exhibited significantly greater respiratory depression. Poincaré analyses show that GS/GS mice have diminished respiratory variability compared with WT mice. Modulation of G(alpha)(i2) signaling by RGS proteins alters loss and resumption of wakefulness and state-dependent changes in breathing.
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    ABSTRACT: The Regulator of G protein Signaling (RGS) proteins were identified as a family in 1996 and humans have more than 30 such proteins. Their best known function is to suppress G Protein-Coupled Receptors (GPCR) signaling by increasing the rate of Gα turnoff through stimulation of GTPase activity (i.e., GTPase acceleration protein or GAP activity). The GAP activity of RGS proteins on the Gαi and Gαq family of G proteins can terminate signals initiated by both α and βγ subunits. RGS proteins also serve as scaffolds, assembling signal-regulating modules. Understanding the physiological roles of RGS proteins is of great importance, as GPCRs are major targets for drug development. The traditional method of using RGS knockout mice has provided some information about the role of RGS proteins but in many cases effects are modest, perhaps because of redundancy in RGS protein function. As an alternative approach, we have utilized a glycine-to-serine mutation in the switch 1 region of Gα subunits that prevents RGS binding. The mutation has no known effects on Gα binding to receptor, Gβγ, or effectors. Alterations in function resulting from the G>S mutation imply a role for both the specific mutated Gα subunit and its regulation by RGS protein activity. Mutant rodents expressing these G>S mutant Gα subunits have strong phenotypes and provide important information about specific physiological functions of Gαi2 and Gαo and their control by RGS. The conceptual framework behind this approach and a summary of recent results is presented in this chapter.
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