Interestingly, the basic EC virodhamine (VIR), which possesses an amino group instead of a hydroxyl or carboxyl group, did not significantly alter the a2 and a3 GlyR responses and behaved as a very weak a1 GlyR modulator (Figure 1A-C). These results suggest that hydroxyl groups on ECs are required for the positive modulation of all three GlyR isoforms, whereas carboxyl groups are structural prerequisites for the inhibition of a2 and a3 GlyRs. These findings strongly suggest that the existence of specific molecular sites in the different GlyR isoforms which underlie subunit-specific actions of ECs on the GlyR subtypes. We next aimed at determining the molecular mechanisms and elements underlying the differential Abmole Axitinib allosteric modulation of GlyRs by acidic ECs. To this end, we focused on NA-Gly and examined its actions at different glycine concentrations (Figure S2). Interestingly, the potentiation elicited by NA-Gly on a1 GlyRs at EC10 of glycine was significantly attenuated at a higher glycine concentration (EC50), whereas the NA-Gly-induced inhibition of a2 and a3 GlyRs remained unaltered. These results suggest that the mechanisms and molecular sites involved in potentiation and inhibition are different and non-conserved between subunits. In order to define the molecular sites involved, we next examined the NA-Gly effects using a set of chimeric and mutant GlyRs. To analyze the importance of TM4 and the IL domains, we first studied a pair of chimeric GlyR constructs in which the regions upstream of the IL between a1 and a2 were exchanged (Figure 2A). We found that this exchange did not significantly Gefitinib EGFR/HER2 inhibitor affect the modulation by NA-Gly (Figure 2B-C). Based on these results, we can conclude that IL and TM4 domains do not significantly contribute to the subunit-specific modulation by NA-Gly. Next, we evaluated the importance of TM regions upstream from the IL. Previous studies have shown that TMregions of GABAA and GlyRs contain critical residues for several allosteric modulators, such as ethanol, general anesthetics and neurosteroids. Furthermore, very recent evidences have been reported that TM residues are critical for the potentiation of recombinant a1 GlyRs by D9-tetrahydrocannabinol (THC) and cannabidiol, two phytocannabinoids.