Structure of an agonist-bound ionotropic glutamate receptor

Abstract

Ionotropic glutamate receptors (iGluRs) mediate most excitatory neurotransmission in the central nervous system and function by opening their ion channel in response to binding of agonist glutamate. Here, we report a structure of a homotetrameric rat GluA2 receptor in complex with partial agonist (S)-5-nitrowillardiine. Comparison of this structure with the closed-state structure in complex with competitive antagonist ZK 200775 suggests conformational changes that occur during iGluR gating. Guided by the structures, we engineered disulfide cross-links to probe domain interactions that are important for iGluR gating events. The combination of structural information, kinetic modeling, and biochemical and electrophysiological experiments provides insight into the mechanism of iGluR gating.

Fig. 1. Functional characterization and structure of NOW-bound GluA2*

(A) Representative whole-cell currents recorded at −60 mV membrane potential from HEK 293 cells expressing GluA2* in response to a 500 ms application of 1 mM Glu alone, application of Glu in the continuous presence of 30 μM of the positive allosteric modulator cyclothiazide (CTZ), application of 100 μM NOW alone and application of NOW in the continuous presence of CTZ. Filled and open triangles indicate steady state currents at the end of NOW and Glu applications, respectively. (B) Occupancies of the closed (C), open (O) and desensitized (D) states of the receptor at the time points indicated by the triangles in (A). Subscript `A' and `*' indicate agonist bound and ion conducting states, respectively. The occupancies are predicted using kinetic modeling (Fig. S2). (C) The 'broad' (left) and 'narrow' (right) faces of the GluA2_NOW structure viewed parallel to the membrane and perpendicular to the overall 2-fold axis of molecular symmetry. Inner and outer sides of membrane are indicated by parallel grey bars. Each of four subunits is in different color.

Fig. 2. Comparison of NOW-bound and ZK-bound GluA2*

(A) Superposition of the full length GluA2_NOW (red) and GluA2_ZK (blue). Red arrows show changes in the GluA2_NOW structure compared to GluA2_ZK. (B) Superposition of individual LBDs from GluA2_NOW and GluA2_ZK structures based on the upper lobe D1 and viewed along the axis of 11° rotation that brings the lower lobe D2 of GluA2_ZK into GluA2_NOW. (C–D) LBD dimers from the GluA2_ZK (C) and GluA2_NOW (D) structures. Shown are Cα's (yellow spheres) and distances between them for E487 and I633 as well as ZK and NOW molecules as stick models.

Fig. 3. LBD dimer interface crosslinking and high mobility of LBD

(A–B) Superposition of LBD dimers from GluA2_NOW (red) and GluA2_ZK (blue) structures based on the upper lobes D1 and viewed perpendicular to (A) or along (B) the axes of rotation that brings the lower lobes D2 of GluA2_ZK into GluA2_NOW. Spheres show Cα's for residues at the LBD interface substituted with cysteines. (C) Table showing LBD cross-interface distances between Cα's of the cysteine substituted residues. Each number is the average of distances for the AD and BC dimers in Angstroms. (D) SDS-PAGE analysis of spontaneous crosslinking of cysteines introduced at the LBD interface. The experiments were carried out with GluA2* receptors either in reducing conditions (2 mM DTT, left lanes) or in non-reducing conditions but in the presence of 3 mM Glu and 50 μM CTZ (Glu+CTZ, favoring the open state) or 3 mM Glu (Glu, favoring the desensitized state) or 500 μM NOW (NOW, favoring the desensitized state) or 100 μM ZK (ZK, favoring the antagonist bound closed state) or in the absence of ligands (Apo, favoring the unliganded closed state). Filled and open triangles indicate positions of monomeric and dimeric bands, respectively. Note, in the absence of reducing agent, substituted cysteines can crosslink in conditions that favor different gating states, indicating high mobility of GluA2* LBDs in solution.

Fig. 4. Effects of LBD dimer interface crosslinking on gating

(A) Representative whole-cell currents recorded at −60 mV membrane potential from HEK 293 cell expressing GluA2-K493C in response to a 500 ms application of 1 mM Glu alone and applications of Glu in the continuous presence of 30 μM CTZ or 2 mM DTT or CTZ and DTT. (B) Effects of cysteine crosslinking on activation. Shown is the ratio of initial currents recorded in the continuous presence of CTZ in the absence and presence of DTT. (C) Effects of cysteine crosslinking on GluA2 desensitization. Shown is the extent of current reduction due to desensitization measured in the absence (filled bars) or presence (open bars) of DTT.