Modulatory Actions of the Glycine Receptor β Subunit on the Positive Allosteric Modulation of Ethanol in α2 Containing Receptors.

Braulio Muñoz,Gustavo Moraga-Cid,Luis G Aguayo,Trinidad Mariqueo,Gonzalo E Yevenes,Pablo Murath,Christian Peters,Robert W Peoples

doi:10.3389/fnmol.2021.763868

Braulio Muñoz, Gustavo Moraga-Cid + Show 6 more

Open Access

https://doi.org/10.3389/fnmol.2021.763868

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Abstract

Alpha1-containing glycine receptors (GlyRs) are major mediators of synaptic inhibition in the spinal cord and brain stem. Recent studies reported the presence of α2-containing GlyRs in other brain regions, such as nucleus accumbens and cerebral cortex. GlyR activation decreases neuronal excitability associated with sensorial information, motor control, and respiratory functions; all of which are significantly altered during ethanol intoxication. We evaluated the role of β GlyR subunits and of two basic amino acid residues, K389 and R390, located in the large intracellular loop (IL) of the α2 GlyR subunit, which are important for binding and functional modulation by Gβγ, the dimer of the trimeric G protein conformation, using HEK-293 transfected cells combined with patch clamp electrophysiology. We demonstrate a new modulatory role of the β subunit on ethanol sensitivity of α2 subunits. Specifically, we found a differential allosteric modulation in homomeric α2 GlyRs compared with the α2β heteromeric conformation. Indeed, while α2 was insensitive, α2β GlyRs were substantially potentiated by ethanol, GTP-γ-S, propofol, Zn2+ and trichloroethanol. Furthermore, a Gβγ scavenger (ct-GRK2) selectively attenuated the effects of ethanol on recombinant α2β GlyRs. Mutations in an α2 GlyR co-expressed with the β subunit (α2AAβ) specifically blocked ethanol sensitivity, but not propofol potentiation. These results show a selective mechanism for low ethanol concentration effects on homomeric and heteromeric conformations of α2 GlyRs and provide a new mechanism for ethanol pharmacology, which is relevant to upper brain regions where α2 GlyRs are abundantly expressed.

Highlights

Alcohol use disorder and alcoholism are major health problems affecting millions of people worldwide and causing great medical and economic burdens
Glycine receptors can be expressed in recombinant systems as homomeric or heteromeric complexes (4 α subunits:1 β subunit) (Yu et al, 2021; Zhu and Gouaux, 2021) and their expression can be monitored looking at changes on their properties such as time to activation and glycine affinity (Figure 1A)
Because the WT subunits display two basic residues in the intracellular loop (IL) that are important for ethanol modulation (Yevenes et al, 2010; Munoz et al, 2020), we replaced the K389 and R390 residues in the WT α2 glycine receptors (GlyRs) (α2AAβ) to test their role in the heteropentameric receptor (Gallegos et al, 2021)

Summary

Introduction

Alcohol use disorder and alcoholism are major health problems affecting millions of people worldwide and causing great medical and economic burdens. Inhibitory glycine receptors (GlyRs) play a central role controlling spinal and brain stem excitability β GlyR Subunit in Ethanol Modulation (Legendre, 2001; Harvey et al, 2004; Lynch, 2004), and it is widely accepted that pharmacologically relevant concentrations of ethanol positively modulate α1 containing GlyRs (Aguayo and Pancetti, 1994; Eggers et al, 2000; Sebe et al, 2003). Most studies that have examined the effects of ethanol on recombinant GlyRs have used homomeric conformations of α1 or α2 expressed in HEK 293 cells or oocytes (Crawford et al, 2008; Yevenes et al, 2010; McCracken et al, 2013). The studies showed that the α2 subunit was less sensitive to ethanol than α1 homomeric subunits (Yevenes et al, 2010). These studies indicated that the intracellular loop (IL) molecular requirements are present in the α2 subunit, the channel is not a target for positive allosteric modulation by ethanol (Yevenes et al, 2010)

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