Glycine Transporter 2: Mechanism and Allosteric Modulation.
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ABSTRACT:
SUBMITTER: Frangos ZJ
PROVIDER: S-EPMC8602798 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol sensitivity of diverse GlyR isoforms and mutants was explained by the presence of specific residues in putative alcohol pockets. Here, we demonstrate that ethanol sensitivity in two ligand-gated ion receptor members, the GlyR adult ?(1) and embryonic ?(2) subunits, can be modified through selective mutations that rescued or impaired G?? modulation. Even though both isoforms were able to physically interact with G??, only the ?(1) GlyR was functionally modulated by G?? and pharmacological ethanol concentrations. Remarkably, the simultaneous switching of two transmembrane and a single extracellular residue in ?(2) GlyRs was enough to generate GlyRs modulated by G?? and low ethanol concentrations. Interestingly, although we found that these TM residues were different to those in the alcohol binding site, the extracellular residue was recently implicated in conformational changes important to generate a pre-open-activated state that precedes ion channel gating. Thus, these results support the idea that the differential ethanol sensitivity of these two GlyR isoforms rests on conformational changes in transmembrane and extracellular residues within the ion channel structure rather than in differences in alcohol binding pockets. Our results describe the molecular basis for the differential ethanol sensitivity of two ligand-gated ion receptor members based on selective G?? modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs.
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Project description:Glycine receptors (GlyRs) are transmitter-gated anion channels of the Cys-loop superfamily which mediate synaptic inhibition at spinal and selected supraspinal sites. Although they serve pivotal functions in motor control and sensory processing, they have yet to be exploited as drug targets partly because of hitherto limited possibilities for allosteric control. Endocannabinoids (ECs) have recently been characterized as direct allosteric GlyR modulators, but the underlying molecular sites have remained unknown. Here, we show that chemically neutral ECs (e.g. anandamide, AEA) are positive modulators of ?(1), ?(2) and ?(3) GlyRs, whereas acidic ECs (e.g. N-arachidonoyl-glycine; NA-Gly) potentiate ?(1) GlyRs but inhibit ?(2) and ?(3). This subunit-specificity allowed us to identify the underlying molecular sites through analysis of chimeric and mutant receptors. We found that alanine 52 in extracellular loop 2, glycine 254 in transmembrane (TM) region 2 and intracellular lysine 385 determine the positive modulation of ?(1) GlyRs by NA-Gly. Successive substitution of non-conserved extracellular and TM residues in ?(2) converted NA-Gly-mediated inhibition into potentiation. Conversely, mutation of the conserved lysine within the intracellular loop between TM3 and TM4 attenuated NA-Gly-mediated potentiation of ?(1) GlyRs, without affecting inhibition of ?(2) and ?(3). Notably, this mutation reduced modulation by AEA of all three GlyRs. These results define molecular sites for allosteric control of GlyRs by ECs and reveal an unrecognized function for the TM3-4 intracellular loop in the allosteric modulation of Cys-loop ion channels. The identification of these sites may help to understand the physiological role of this modulation and facilitate the development of novel therapeutic approaches to diseases such as spasticity, startle disease and possibly chronic pain.
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