Activated G Protein α_sSubunits Increase the Ethanol Sensitivity of Human Glycine Receptors

Abstract

It is well known that ethanol modulates the function of the Cys loop ligand-gated ion channels, which include the inhibitory glycine receptors (GlyRs). Previous studies have consistently shown that transmembrane and extracellular sites are essential for ethanol actions in GlyRs. In addition, recent evidence has shown that the ethanol modulation of GlyRs is also affected by G protein activation through G␤␥ subunits. However, more specific roles of G protein ␣ subunits on ethanol actions are unknown. Here, we show that the allosteric effect of ethanol on the human ␣ 1 GlyR is selectively enhanced by the expression of G␣ s Q-L. For example, constitutively active G␣ s , but not G␣ q or G␣ i , was able to displace the alcohol sensitivity of GlyRs toward low millimolar concentrations (17 Ϯ 4 versus 48 Ϯ 5% at 100 mM). Experiments under conditions that increased cAMP and protein kinase A (PKA)-mediated signaling, on the contrary, did not produce the same enhancement in sensitivity, suggesting that the G␣ s Q-L effect was not dependent on cAMP/PKAdependent signaling. On the other hand, the effect of G␣ s Q-L was blocked by a G␤␥ scavenger (9 Ϯ 3% of control). Furthermore, two mutant receptors previously shown to have impaired interactions with G␤␥ were not affected by G␣ s Q-L, suggesting that G␤␥ is needed for enhancing ethanol sensitivity. These results support the conclusion that activated G␣ s can facilitate the G␤␥ interaction with GlyRs in presence of ethanol, independent of increases in cAMP signaling. Thus, these data indicate that the activated form of G␣ s is able to positively influence the effect of ethanol on a type of inhibitory receptor important for motor control, pain, and respiration.

Intragenic suppression of a constitutively active allele of Gsα associated with McCune–Albright syndrome

“…Nearby residues in switch II, namely G226 (Gao 1995) and Q227 (Masters 1989), are well-established residues that, when mutated, can abolish GTPase activity of the alpha subunit, rendering it constitutively active. Homologous mutations to Q227 in many other alpha subtypes have been shown to be constitutively active, and have been used in numerous studies of the role of alpha subunits in diverse cell signaling pathways (for example, Jeon 2012, Yevenes 2011, Ogata 2007, Slessareva 2006, Lo 2003). …”

Ethanol effects on glycinergic transmission: From molecular pharmacology to behavior responses

“…Ethanol effects can also be inhibited by overexpression of Gβγ scavengers like ct-GRK2 and ct-GRK3, Gα o or an antibody against Gβ subunits, confirming the involvement of G protein signaling in potentiation of glycine-evoked currents by low ethanol concentrations in α1 GlyRs [148,153]. …”

Titrating Tipsy Targets: The Neurobiology of Low-Dose Alcohol

“…Amino acid residues in the N-terminal domain (Ala52) and in TM1 – 4 are crucial for alcohol sensitivity and may contribute to the actions of alcohol on GlyRs [61, 62]. In addition, the intracellular loop domain (316RFRRK320,385KK386) that mediates G-protein modulation of GlyRs through Gβγ may be responsible for the low-dose alcohol sensitivity of α1 containing GlyRs [59, 63]. The role of G proteins and other factors, such as Zn 2+ + [64], in modulating alcohol sensitivity of GlyRs suggests that intracellular signaling and extracellular environments have a significant impact on the degree of receptor modulation by alcohol.…”