Regulator of G Protein Signaling Protein Suppression of Gα_o Protein-Mediated α_2A Adrenergic Receptor Inhibition of Mouse Hippocampal CA3 Epileptiform Activity

Abstract

Activation of G protein-coupled ␣ 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 ␣ 2 ARs and G proteins (G␣ o or G␣ 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: (Ϫ)EPI Ͼ (Ϫ)NE ϾϾϾ (ϩ)NE. To identify the ␣ 2 AR subtype involved, equilibrium dissociation constants (pK b ) were determined for the selective ␣AR 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 ␣ 2A AR subtype (r ϭ 0.98, slope ϭ 1.07). Furthermore, the inhibitory effects of EPI were lost in hippocampal slices from ␣ 2A AR-but not ␣ 2C AR-knockout mice. Pretreatment with pertussis toxin also reduced the EPImediated inhibition of epileptiform bursts. Finally, using knock-in mice with point mutations that disrupt regulator of G protein signaling (RGS) binding to G␣ subunits to enhance signaling by that G protein, the EPI-mediated inhibition of bursts was significantly more potent in slices from RGS-insensitive G␣ o G184S heterozygous (G␣ o ϩ/GS) mice compared with either G␣ i2 G184S heterozygous (G␣ i2 ϩ/GS) or control mice (EC 50 ϭ 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 ␣ 2A AR/G␣ o protein-mediated pathway under strong inhibitory control by RGS proteins. This suggests a possible role for RGS inhibitors or selective ␣ 2A AR agonists as a novel antiepileptic drug therapy.

A central role for R7bp in the regulation of itch sensation

“…We therefore tested pruriception in mice heterozygous for the G184S mutant of Gαo ( Gnao1 G184S/+) [8]. This approach afforded the opportunity to test the hypothesis without any knowledge regarding the upstream Gi/o-coupled GPCR(s) that might be involved in R7bp-dependent pruriception.…”

“…Kappa-opioid receptor ( Oprk1 ) knockout mice (B6.129S2-Oprk1 tm1Kff /J; Stock Number 007558) were obtained from The Jackson Laboratory (Bar Harbor, Maine). The generation and characterization of mice harboring a heterozygous gain-of-function knock-in mutation in Gnao1 that prevents Gαo turnoff by regulators of G protein signaling proteins ( Gnao1 G184S/+) were previously described [8; 18]. For this study Gnao1 G184S/+ heterozygotes in a C57BL/6 background were employed.…”

Phenotypic spectrum of GNAO1 variants: epileptic encephalopathy to involuntary movements with severe developmental delay

“…Gα o -deficient mice (Gnao1 − / − ) showed occasional seizures and generalized tremor with postnatal death, 15,16 and gainof-function knock-in mutant mice (Gnao1 G184S/+ ) also showed rare seizures, postnatal death, and a markedly increased frequency of interictal epileptiform discharges, 17,18 supporting that seizures and involuntary movement are the two major characteristic features caused by GNAO1 variants. However, no sex differences have been reported in these mouse models, and postnatal death was not influenced by sex in Gnao1 G184S/+ mice.…”

Regulators of G-Protein Signaling and Their Gα Substrates: Promises and Challenges in Their Use as Drug Discovery Targets

“…This "RGS-insensitivity" point mutation (glycine to serine) was originally identified by DiBello et al (1998) in GPA1, the G␣ subunit of the yeast S. cerevisiae, functions equivalently in mammalian G␣ subunits such as G␣ i1 , G␣ o , and G␣ q (Lan et al, 1998;Clark and Traynor, 2004) and has also been shown to leave all other functions of G␣ intact, including intrinsic nucleotide binding and hydrolysis activities, as well as coupling to G␤␥, receptor, and effectors (Lan et al, 1998;Chen et al, 2004;Day et al, 2004;Fu et al, 2004;Ikeda and Jeong, 2004). Separate mouse strains bearing this RGS-insensitivity point mutation (G184S) within their G␣ o or G␣ i2 gene loci, respectively, have been generated (Fu et al, 2004;Huang et al, 2006); these mice possess select changes in various organ system functions controlled by GPCR signaling, including central nervous system, cardiovascular, and endocrine functions (Fu et al, , 2007Huang et al, 2006Huang et al, , 2008Goldenstein et al, 2009;Icaza et al, 2009;Signarvic et al, 2010;Talbot et al, 2010). The most recent findings of Ruiz de Azua et al (2010), that RGS4 is a negative regulator of M3 mAChR signaling to insulin secretion in pancreatic ␤-cells, articulate well with the findings of Huang et al (2008) that RGS-insensitive G␣ i2 (G184S) knock-in mice exhibit increased glucose tolerance when on a high-fat diet.…”