A selective Gβγ-linked intracellular mechanism for modulation of a ligand-gated ion channel by ethanol

Abstract

The current understanding about ethanol effects on the ligand-gated ion channel (LGIC) superfamily has been restricted to identify potential binding sites within transmembrane (TM) domains in the Cys-loop family. Here, we demonstrate a key role of the TM3-4 intracellular loop and G␤␥ signaling for potentiation of glycine receptors (GlyRs) by ethanol. We discovered 2 motifs within the large intracellular loop of the GlyR ␣1 subunit that are critical for the actions of pharmacological concentrations of ethanol. Significantly, the sites were ethanol-specific because they did not alter the sensitivity to general anesthetics, neurosteroids, or longer n-alcohols. Furthermore, G␤␥ scavengers selectively attenuated the ethanol effects on recombinant and native neuronal GlyRs. These results show a selective mechanism for low-ethanol concentration effects on the GlyR and provide a mechanism on ethanol pharmacology, which may be applicable to other LGIC members. Moreover, these data provide an opportunity to develop new genetically modified animal models and novel drugs to treat alcohol-related medical concerns.pharmacology ͉ signal transduction ͉ glycine receptor ͉ alcoholism ͉ G proteins

Inhibition of the Ethanol-induced Potentiation of α1 Glycine Receptor by a Small Peptide That Interferes with Gβγ Binding

“…2). These results demonstrate that there is a region in the IL that interacts with G␤␥ under in vitro conditions and correlates well with previous functional studies (21).…”

“…4). In the control condition, ethanol potentiated the GlyR current to 47 Ϯ 2% above control, in agreement with previous reports (6,20,21). However, use of RQH, RQH C10 , and RQH C7 peptides reduced the potentiation to 19 Ϯ 4%, 17 Ϯ 1%, and 16 Ϯ 4% of control, respectively.…”

“…For example, it was found that a stretch of residues located in the N-terminal region of this domain is important for the interaction with G␤␥ (20). In addition, electrophysiological studies demonstrated that both GTP␥S and ethanol potentiation of GlyR were affected by the mutation of residues 316 -320 pres-ent in this domain (21). The importance of these residues was also shown by direct in vitro interaction between the GlyR-IL and G␤␥ using GST pulldown experiments (20).…”

“…Regulation by signaling pathways has been described for several members of LGICs, including the effects of ethanol and G proteins on GlyRs (6,21,25). In the case of GlyR, it has been previously described that the GlyR-IL contains motifs that are important for G protein-and ethanol-mediated potentiation.…”

“…Baclofen (10 M) was applied in short pulses (4 -5 s) every 2 min during 8 min. For the recording of ethanol-mediated potentiation of GlyR, a previously described methodology was used (6,21). Ethanol (100 mM) was co-applied with glycine (15 M), and the results were expressed as percentage of potentiation at 15 min.…”