Aminomethanesulfonic acid illuminates the boundary between full and partial agonists of the pentameric glycine receptor

Abstract

To clarify the determinants of agonist efficacy in pentameric ligand-gated ion channels, we examined a new compound, aminomethanesulfonic acid (AMS), a molecule intermediate in structure between glycine and taurine. Despite wide availability, to date there are no reports of AMS action on glycine receptors, perhaps because AMS is unstable at physiological pH. Here, we show that at pH 5, AMS is an efficacious agonist, eliciting in zebrafish α1 glycine receptors a maximum single-channel open probability of 0.85, much greater than that of β-alanine (0.54) or taurine (0.12), and second only to that of glycine itself (0.96). Thermodynamic cycle analysis of the efficacy of these closely related agonists shows supra-additive interaction between changes in the length of the agonist molecule and the size of the anionic moiety. Single particle cryo-electron microscopy structures of AMS-bound glycine receptors show that the AMS-bound agonist pocket is as compact as with glycine, and three-dimensional classification demonstrates that the channel populates the open and the desensitized states, like glycine, but not the closed intermediate state associated with the weaker partial agonists, β-alanine and taurine. Because AMS is on the cusp between full and partial agonists, it provides a new tool to help us understand agonist action in the pentameric superfamily of ligand-gated ion channels.

Keywords: agonists; cryoEM; glycine receptors; molecular biophysics; single channel recording; structural biology; zebrafish.

Figure 1.. AMS is a highly efficacious agonist on zebrafish α1 GlyR.

(A) Structures of glycine, β-alanine, AMS, and taurine. (B) Whole-cell current responses of HEK293 cells elicited by application of agonist solutions (pH 5) with a U-tube. Cells were held at –40 mV. (C) Average concentration-response curves for glycine (black), β-alanine (green), AMS (blue), and taurine (red), n=6–9 cells. Responses of AMS, β-alanine, and taurine are normalized to those to a saturating concentration of glycine (100 mM) in each cell. AMS, aminomethanesulfonic acid.

Figure 2.. Maximum open probability evoked by different GlyR agonists.

(A, B) Representative single-channel current recordings of zebrafish α1 GlyR activity evoked by high concentrations of agonists. Recordings were made in the cell attached configuration at +100 mV holding potential. (C) Boxplots of maximum Popen values for clusters with the different agonists (one point per cluster). Boxes and whiskers show the 25th and 75th and the 5th and 95th percentiles, respectively. The horizontal black line in the box is the median.

Figure 3.. Cryo-EM analysis of structures of zebrafish α1 GlyR bound to AMS.

(A–C) Cryo-EM density maps for desensitized, open, and expanded-open states with one subunit highlighted. The AMS density is in red. (D–F) Atomic models for desensitized, open, and expanded-open states. Shown are GlyR in cartoon representation, AMS in sphere representation (red), and N-glycans in stick representation. AMS, aminomethanesulfonic acid; EM, electron microscopy.

Figure 3—figure supplement 1.. GlyR purification.

(A) SEC trace for purification of GlyR. (B) SDS-PAGE analysis for peak fraction.

Figure 3—figure supplement 2.. Flow chart for cryo-EM data processing of GlyR bound with AMS.

Figure 3—figure supplement 3.. 3D reconstruction of GlyR-AMS states.

(A, C, E) Local resolution maps for open (A), desensitized (C), and expanded-open states (E), respectively. (B, D, F) FSC curves without (orange) and with (red) mask generated by cryoSparc, and between the model and the final maps for open (B), desensitized (D), and expanded-open states (F), respectively.

Figure 4.. Comparison of ion channel pores.

(A, B) Shape and ion permeation pathway for AMS-bound desensitized (see also (C)) and open (see also (D)) states. M2 helices and key amino acids are shown in ribbon and stick representation, respectively. Purple, green, and red spheres define radii >3.5 Å, 1.8–3.5 Å, and <1.8 Å. (C, D) Profiles of pore radii calculated by the HOLE program for desensitized (A) and open (B) states bound with AMS, taurine, and glycine. The Cα position of R268 was set to 0. AMS, aminomethanesulfonic acid.

Figure 5.. Comparison of agonist binding sites.

(A) Two adjacent GlyR subunits are shown in cartoon representation. The agonist binding pocket is indicated by a black box. (B, C) Stereo figures of the binding sites showing likely hydrogen and cation-π interactions with AMS (B) and glycine (C) bound, respectively. Numbers are the distances in Å of probable cation- π interactions. Numbering of residues includes the signal peptide of 16 amino acids. (D) Comparison of the positions of key binding residues in the open states of the glycine (salmon), taurine (green), and AMS (blue) complexes, obtained by superposing the respective ECDs. (E) Schematic diagram illustrating the distances (Å) between the Cα atoms of key amino acids in glycine-, taurine-, and AMS-bound open states. AMS, aminomethanesulfonic acid; ECD, extracellular domain.

Figure 5—figure supplement 1.. Comparison of loops B and C.

(A) Density of AMS contoured at 8.5σ in the GlyR open state. For AMS, carbon, sulfur, and nitrogen atoms are colored in blue, yellow, and red, respectively. The position of the agonist binding pocket is shown by the box in the subunit dimer shown in cartoon representation on the left. (B) Conformation of the taurine binding site showing likely hydrogen and cation-π interactions with the agonist. Distances of cation-π interaction shown in Å. The color coding is the same to panel (A). The gray spheres represent the centers of mass of the benzene ring. (C) Comparison of the secondary elements loops B and C in the binding pockets for glycine- (salmon), taurine- (green), and AMS-bound (blue) open states by superposing the ECD of the (−) subunit. AMS, aminomethanesulfonic acid; ECD, extracellular domain.

Figure 6.. Comparison of the ECD-TMD interface in different agonist-bound complexes.

(A) Superposition of the ECD-TMD interface of the open states of the glycine (salmon), taurine (green), or AMS (blue) bound forms. The key amino acids at the ECD-TMD interface are shown in stick representation. Key secondary structure elements are labelled. The blue spheres represent the centers of mass of the secondary structure elements for the AMS-bound structure. (B) Schematic diagram illustrating the distances (Å) of the center of mass points shown in panel (A) of glycine-, taurine-, and AMS-bound open states. ECD, extracellular domain; TMD, transmembrane domain.

Figure 7.. Thermodynamic cycle for the four GlyR agonists functionally characterized, showing their structure and example sweeps of the single-channel activity they elicit.

Author response image 1.

Author response image 2.