Kumar A et al. (2020), Mechanisms of activation and desensitization of...

XB-ART-57341

Nat Commun 2020 Jul 27;111:3752. doi: 10.1038/s41467-020-17364-5.

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

Kumar A , Basak S , Rao S , Gicheru Y , Mayer ML , Sansom MSP , Chakrapani S .

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Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structures have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unanswered. Here, we present Cryo-EM structures of the full-length GlyR protein complex reconstituted into lipid nanodiscs that are captured in the unliganded (closed), glycine-bound (open and desensitized), and allosteric modulator-bound conformations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation. The functional state assignments were validated by molecular dynamics simulations, and the observed permeation events are in agreement with the anion selectivity and conductance of GlyR. These studies provide the structural basis for gating, ion selectivity, and single-channel conductance properties of GlyR in a lipid environment.

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Genes referenced: ecd ttn

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	Fig. 1. Cryo-EM structures of full-length GlyR in multiple conformational states.a Ion permeation pathway generated with HOLE60 for GlyRâApo (salmon red), GlyRâGly/PTX (deep teal) and GlyRâGly (slate blue). For clarity, the cartoon representation of only two non-adjacent subunits are shown. Green and purple spheres define radii of 1.8â3.3âÃ and >3.3âÃ, respectively. The residues located at various pore constrictions are shown as sticks. b The pore radius is plotted as a function of distance along the pore axis. The dotted line indicates the approximate radius of a hydrated chloride ion, which is estimated at 2.26âÃ. c A view of M2 helices from the extracellular end for the three GlyR conformations. Positions Leu9â² and Pro-2â² are shown in ball-and-stick representation and the corresponding distances between CÎ± are given in Ã.
	Fig. 2. Conformational changes underlying GlyR gating.a Cryo-EM density segments for neurotransmitter binding site residues and glycine ligand as seen in the GlyRâGly structure. Residues in the principal subunit and complementary subunit are indicated in black and magenta, respectively. b LigPlot analysis of glycine orientation in the binding pocket and residues within 4âÃ distance are displayed in red70. c Comparison of the neurotransmitter binding site for the GlyRâApo and GlyRâGly conformations. The residues that are involved in neurotransmitter binding are shown in sticks. d Cryo-EM map showing the density for M2 and PTX bound in the pore of GlyRâGly/PTX. The interacting residues are shown in stick representation. For clarity, M2 for only two diagonal subunits are shown. e LigPlot analysis of PTX and the interacting residues. f A close-up of the M2 conformations is shown upon aligning the three GlyR conformations. Positions Leu9â² and Pro-2â² are shown as sticks.
	Fig. 3. Global conformational changes and altered inter-domain interactions during GlyR gating.a A side-view of the ECD interface in GlyRâ€“Apo and GlyRâ€“Gly. The principal subunits are colored (GlyRâ€“Apo: salmon red and GlyRâ€“Gly: slate blue) while the complementary subunits are shown in shades of gray. b A side view of the TMD in GlyRâ€“Apo and GlyRâ€“Gly conformations. c Interactions at the subunit- and domain-interfaces between ECDâ€“ECD, ECDâ€“TMD, and TMDâ€“TMD in GlyRâ€“Apo (left) and GlyRâ€“Gly (right). The residues involved in the interactions are shown in sticks and interactions are highlighted by black dashes. Mutations at positions shown in green are associated with hyperekplexia2.
	Fig. 4. Conformational changes in the M4 helix and the effects on internal cavities.a An overlay of GlyRâ€“Apo and GlyRâ€“Gly. The TMD viewed from the top reveals repositioning of TM helices. The intra-subunit and inter-subunit cavities are indicated by green and gray arrows, respectively. b Extent of conformational change in the ICD formed by the pre-M4 region, the M1â€“M2 linker, and the post-M3 region. The direction of movement is highlighted by black arrows. c A comparison of the inter-subunit (gray dots) and intra-subunit (green dots) cavities in GlyRâ€“Apo (top) and GlyRâ€“Gly (bottom) predicted using F pocket algorithm61. The cavities predicted on the surface were removed for clarity. The right and left panels are side and top views, respectively. The residues shown in sticks on the left panel are implicated in binding allosteric ligands45.
	Fig. 5. Analysis of GlyRâ€“Gly/IVM structures.a TEVC recording of 1â€‰mM glycine-induced currents in the presence and absence of 0.5â€‰Î¼M IVM (left). Representative trace from multiple independent oocyte recordings (nâ€‰=â€‰5). The traces are normalized to the peak current to show the current decay. Ion permeation pathway for GlyRâ€“Gly/IVM-1 GlyRâ€“Gly/IVM-2 (right). Green and purple spheres define radii of 1.8â€“3.3â€‰Ã… and >3.3â€‰Ã…, respectively. The residues located at various pore constrictions are shown as sticks. b Current recording for 0.1â€‰mM glycine and 0.1â€‰mM glycine and 30â€‰Î¼M IVM (left). Representative trace from multiple independent oocyte recordings (nâ€‰=â€‰3). The pore profile for GlyRâ€“Gly/IVM-3 structure. c A view of M2 helices from the extracellular end Positions Leu9â€² and Pro-2â€² and the corresponding distances between CÎ± in Ã…. d Alignment of GlyR structures showing the conformational differences at the level of Pro-2â€². Dotted arrows indicate the direction of rotation going from the GlyRâ€“Apo (closed) to the GlyRâ€“Gly (desensitized) state.
	Fig. 6. Molecular dynamics simulations of GlyR conformations.a Mean pore radius profiles and standard deviations averaged across three independent 30âns equilibrium simulations for GlyRâApo (left), GlyRâGly/PTX (middle), and GlyRâGly (right) along the central pore axis. The final 20âns of each 30âns simulation trajectory was used to evaluate these profiles. The one-standard-deviation range between calculations (nâ=â3, independent repeats of 30âns simulations) are shown as a gray band and the thick line is the mean between the triplicate 30âns simulations. Major constriction sites are indicated and the dotted line denotes the radius of hydrated chloride ion. The black traces are the pore radius profile calculated from the cryo-EM structures. b Corresponding mean water free energy profiles and standard deviations. Peaks in free energy profiles are highlighted. c Trajectories along the pore (z)-axis of water molecules and chloride ion coordinates within 5âÃ of the channel axis inside the pore, in the presence of a +500âmV transmembrane potential difference (i.e., with the cytoplasmic side having a positive potential). One of five independent 200âns replicates is shown for each structure. During these and the preceding simulations, positional restraints were placed on the protein backbone, in order to preserve the experimental conformational state while permitting rotameric flexibility in amino acid side chains. The energetic barriers due to the ring of Leu9â² and Pro-2â² are at z ~0 and â20âÃ, respectively.

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