Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and gamma-aminobutyric acid type A receptors (GABA(A)Rs). To test this, we mutated Loop 2 in the alpha 1 subunit of GlyRs and in the gamma subunit of alpha 1 beta 2 gamma 2GABA(A)Rs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of alpha 1GlyR subunits with Loop 2 from the delta GABA(A)R (delta L2), but not the gamma GABA(A)R subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the gamma subunit of GABAARs with delta L2 shifted the ethanol threshold from 50 mM in WT to 1 mM in the GABA(A) gamma-delta L2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABAARs. The delta L2 mutations did not affect GlyR or GABA(A)R sensitivity, respectively, to Zn2+ or diazepam, which suggests that these delta L2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and delta L2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.