Correlating efficacy and desensitization with GluK2 ligand-binding domain movements

Naushaba Nayeem,Olga Mayans,Tim Green

doi:10.1098/rsob.130051

Abstract

Gating of AMPA- and kainate-selective ionotropic glutamate receptors can be defined in terms of ligand affinity, efficacy and the rate and extent of desensitization. Crucial insights into all three elements have come from structural studies of the ligand-binding domain (LBD). In particular, binding-cleft closure is associated with efficacy, whereas dissociation of the dimer formed by neighbouring LBDs is linked with desensitization. We have explored these relationships in the kainate-selective subunit GluK2 by studying the effects of mutating two residues (K531 and R775) that form key contacts within the LBD dimer interface, but whose truncation unexpectedly attenuates desensitization. One mutation (K531A) also switches the relative efficacies of glutamate and kainate. LBD crystal structures incorporating these mutations revealed several conformational changes that together explain their phenotypes. K531 truncation results in new dimer contacts, consistent with slower desensitization and sideways movement in the ligand-binding cleft correlating with efficacy. The tested mutants also disrupted anion binding; no chloride was detected in the dimer-interface site, including in R775A where absence of chloride was the only structural change evident. From this, we propose that the charge balance in the GluK2 LBD dimer interface maintains a degree of instability, necessary for rapid and complete desensitization.

Highlights

Both AMPA- and kainate-selective ionotropic glutamate receptors desensitize rapidly and completely in response to glutamate [1]
D776K results in a new, crossdimer interaction [7], but there is currently no structural explanation for the K531 mutant phenotypes. We have addressed this question, characterizing three GluK2 apical mutants with attenuated desensitization (K531A, K531AT779G and R775A) and identifying the conformational changes underlying their functional phenotypes
The anion and cation binding sites formed in the interface between kainate subunit ligand-binding domain (LBD) play an essential role in receptor function [17,29]

Summary

Introduction

Both AMPA- and kainate-selective ionotropic glutamate receptors (iGluRs) desensitize rapidly (with time constants typically approx. 1–10 ms) and completely (by approx. 96–99.8%) in response to glutamate [1]. 96–99.8%) in response to glutamate [1] Desensitization of these ‘non-NMDA’ receptors involves rearrangement of a dimer formed by neighbouring ligand-binding domains (LBDs) [2,3]. Consistent with this, macroscopic desensitization in both AMPA and kainate receptors (KARs) can be blocked by covalently linking (with disulphides) the LBD dimer [5,6]. In both AMPA receptors and KARs, key inter-domain contacts are formed between residues at the edges of D1 (figure 1a, pink surface), and mutations to these edge sites can variously block [9] or attenuate [6,8,10] desensitization

Methods

Results

Discussion

Conclusion