The intracellular domain of homomeric glycine receptors modulates agonist efficacy

Abstract

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for ICD in pentameric ligand-gated channels.

Thermophoretic analysis of ligand-specific conformational states of the inhibitory glycine receptor embedded in copolymer nanodiscs

“…5 c). We speculate that the impaired coupling of the ECD and the TMD is driven (i) by lipid or sterol modulations, as described for other members of pLGICs 40 – 42 and supported by recent findings supposing interactions between glycine receptors and cholesterol 43 or (ii) by the impact of the ICD 44 . This would explain the high variability of aEC 50 values seen upon electrophysiological measurements of GlyRs in the brain and in different heterologous expression systems.…”

“…Thus, the apparent agonist-affinities of the GlyR obtained in electrophysiological measurements are likely determined by conformational transitions after the agonist-induced ECD closure which is in agreement with several postulated intermediate shut states between the resting and open conformation, as indicated by different primed or flipped schemas. Our data may also have some implications for the understanding of the differential impact of (i) mutations 45 and modulators 25 in the ECD and (ii) the TMD and ICD 44 at the GlyR in affecting partial agonism. Therefore mutations and modulators acting in the ECD might affect the conformational change in the ECD layer upon agonist binding whereas modulators acting in the TMD and the ICD may alter intermediate shut states between the resting and open conformation.…”

Mechanisms of activation and desensitization of full-length glycine receptor in lipid nanodiscs

“…The ICD, besides its role in trafficking, has also been implicated to alter single-channel conductance 15 , modulation of gating 16 , and desensitization 17 . In addition, a recent report shows that ICD truncation in GlyR leads to an increase in maximum single-channel open probability and a substantial increase in agonist efficacy 18 . Furthermore, lipids are essential components for membrane protein stability, and it therefore seems likely that both the ICD and lipid environment are important for maintaining the structural integrity of the channel during gating conformational cycles.…”

Markov State Models of Proton- and Gate-Dependent Activation in a Pentameric Ligand-Gated Ion Channel

“…Although P open has not been determined for GLIC, recent cryo-EM structures of closed-state GLIC solved at activating conditions point towards a significant closed population even at activating conditions [26,33]. Other channels in the pLGIC family have also been shown to attain a range of maximal open probabilities; 10-40% for GABA A Rs [36,37], 20-80% for 5-HT 3 s [38,39], 0.2-3% for nAChRs [40,41], and 90-100% for GlyRs [42,43], when saturated with their respective natural agonist. Given the spread in open probability between pLGIC subtypes it seems reasonable that a more distant bacterial homolog like GLIC could have a unique energy landscape.…”