Mechanism of human α3β GlyR modulation in inflammatory pain and 2, 6-DTBP interaction

Abstract

α3β glycine receptor (GlyR) is a subtype of the GlyRs that belongs to the Cys-loop receptor superfamily. It is a target for non-psychoactive pain control drug development due to its high expression in the spinal dorsal horn and indispensable roles in pain sensation. α3β GlyR activity is inhibited by a phosphorylation in the large internal M3/M4 loop of α3 through the prostaglandin E2 (PGE₂) pathway, which can be reverted by a small molecule analgesic, 2, 6-DTBP. However, the mechanism of regulation by phosphorylation or 2, 6-DTBP is unknown. Here we show M3/M4 loop compaction through phosphorylation and 2, 6-DTBP binding, which in turn changes the local environment and rearranges ion conduction pore conformation to modulate α3β GlyR activity. We resolved glycine-bound structures of α3β GlyR with and without phosphorylation, as well as in the presence of 2, 6-DTBP and found no change in functional states upon phosphorylation, but transition to an asymmetric super open pore by 2, 6-DTBP binding. Single-molecule Forster resonance energy transfer (smFRET) experiment shows compaction of M3/M4 loop towards the pore upon phosphorylation, and further compaction by 2, 6-DTBP. Our results reveal a localized interaction model where M3/M4 loop modulate GlyR function through physical proximation. This regulation mechanism should inform on pain medication development targeting GlyRs. Our strategy allowed investigation of how post-translational modification of an unstructured loop modulate channel conduction, which we anticipate will be applicable to intrinsically disordered loops ubiquitously found in ion channels.

Figure 1. Functional characterization of human α3β GlyR.

a, The effect of PGE₂ on glycinergic signaling. PGE₂ activates EP₂ receptors which in turn phosphorylate α3 subunits in a PKA-dependent manner to inhibit Cl⁻ flux through α3β GlyR. b, Typical glycine response of α3wtβwt, α3emβem, α3S346Eβwt and 3S346Eemβem at 30 μM and 2 mM glycine concentration. c, Dose response with Hill fits (lines). Data are represented as mean ± S.E.M. (n=7–11 cells). d, Representative the effect of 10 μM PGE₂ on glycine-evoked current traces in HEK293T cells co-transfected with the α3wtβwt and EP₂ receptor (top), and after disruption of the PKA consensus sequence by introducing the S346A mutation(down). e, Representative the effect of 10 μM PGE₂ on glycine-evoked current traces in HEK293T cells co-transfected with the α3emβem and EP₂ receptor (top), and after introducing the S346A mutation(down). f, Representative traces of glycine-induced whole-cell currents recorded from HEK293T cells expressing α3wtβwt, α3emβem, α3S346Eβwt and 3S346Eemβem GlyRs in the absence or presence of 100 μM 2, 6-DTBP.

Figure 2. Conformational differences between α3β-gly, α3S346Eβ-gly, and α3S346E3-gly/2, 6-DTBP GlyRs.

a, Side view of cryo-EM map of α3β GlyR in digitonin in presence of glycine. d, Side (left) and top-down (right) view of the atomic models. α subunits and β subunit are respectively colored in bright green and yellow. c, Plot of pore radii calculated by the HOLE program for the α3β-gly (green), α3S346Eβ-gly (blue), α3S346Eβ-gly/2, 6-DTBP (orange), α2β-gly (desensitized, PDB ID: 5BKF, green dashed line), and α1β-gly (expanded open, PDB ID:8DN2, red dashed line). d, f, h,Ion permeation pathways for α3β-gly(d), α3S346Eβ-gly(f), and α3S346Eβ-gly/2, 6-DTBP(h) GlyRs. M2 helices are shown as cartoon and the side chains of pore-lining residues as sticks. Purple, green, red spheres define radii of > 3.3 Å, 1.8–3.3 Å, and < 1.8 Å, respectively. e, g, i, Cross-sections of M2 helices at residues 9’ (top) and −2’ (bottom) for α3β-gly(e), α3S346Eβ-gly(g), and α3S346Eβ-gly/2, 6-DTBP(i) GlyRs with distances between neighboring Cα shown in Å.

Figure 3. TMD of α3β and α3S346Eβ GlyRs conformational differences and 2, 6-DTBP induces α3S346Eβ GlyR conformational changes

a, b, c, Z-slice in the TMD at 2’ for α3β-gly (a), α3S346Eβ-gly (b) and α3S346Eβ-gly/2, 6-DTBP(c). d, e, f, Top-down view non-protein densities of α3β-gly (d), α3S346Eβ-gly (e) and α3S346Eβ-gly/2, 6-DTBP (f) models contoured at 5 RMSD. Non-protein densities at widened subunit interfaces, and those in conduction pathways are colored in brown for α3β-gly (d), red for α3S346Eβ-gly (e) and blue for α3S346Eβ-gly/2, 6-DTBP (f), respectively. g, h, i, The cartoon representation of the TMD interfaces between α.C and α. D subunits for α3β-gly (g), α3S346Eβ-gly (h), α3S346Eβ-gly/2, 6-DTBP (i) GlyRs. Red arrow indicates distance between adjacent α subunits at −2’ (down) and 19’(top).

Figure 4. α3S346E mutation and 2, 6-DTBP modulate internal conformation of the M3/M4 loop in α3S346Eβ GlyR

a, Schematic of single-molecule FRET experiments for measuring the FRET value changes of the internal M3/M4 loop induced by α3S346E mutation and 2, 6-DTBP. b, c, Representative single-molecule FRET time traces of α3β(b) and α3S346Eβ(c) GlyRs in apo (top), in presence of glycine (middle) and in presence of glycine and 2, 6-DTBP (bottom). d, e, Histograms of smFRET efficiency values from single-molecules traces for α3β(d) and α3S346Eβ (e) GlyRs in apo (top), in presence of glycine (middle) and in presence of glycine and 2, 6-DTBP (bottom). Two smFRET distributions indicated by curves of Gaussian fitting and the sum of all distributions are shown as black lines.

Figure 5. α3S346E mutation and 2, 6-DTBP affect the distances of M3/M4 loops between different α3 subunits

a, Schematic of single-molecule FRET experiments for measuring the FRET values changes of M3/M4 loops between different α3 subunits. b, c, Representative single-molecule FRET time trace of α3β(b) and α3S346Eβ(c) GlyRs in apo (top), in presence of glycine (middle) and in presence of glycine and 2, 6-DTBP (bottom). d, e, Histograms of smFRET efficiency values from single-molecules traces for α3β(d) and α3S346Eβ(e) GlyR in apo (top), in presence of glycine (middle) and in presence of glycine and 2, 6-DTBP (bottom). Two smFRET distributions indicated by curves of Gaussian fitting and the sum of all distributions are shown as black lines.

Figure 6. Proposed mechanism for α3 M3/M4 loop phosphorylation and 2, 6-DTBP modulation α3β GlyR activity

a, During the entire functional cycle of non-phosphorylated α3β GlyR (close, open, and desensitized states), the M3/M4 loops of α3 subunits are very flexible and unstable. b, Left: Upon α3S346 is phosphorylated, the conformation of M3/M4 loop will change in two aspects: 1) the M3/M4 loop will be folding closer to the TMD; 2) the relative horizontal distances among M3/M4 loops will be fixed. These M3/M4 loops conformational changes result in pore conformational changes to decrease the influx of Cl⁻. Right: 2, 6-DTBP modulate the phosphorylated M3/M4 loop to fold and further closer TMD, leading to pore conformational changes and the phosphorylated α3β GlyR’s activity restoring.