Correlating efficacy and desensitization with GluK2 ligand-binding domain movements

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.

The multifaceted subunit interfaces of ionotropic glutamate receptors

“…While elimination of Na + binding invariably disrupts the dimer, as expected (Plested et al 2008;Nayeem et al 2011), mutations to residues forming the anion binding site have varied functional effects. Most mutations do accelerate the rate of desensitization (Plested & Mayer, 2007;Wong et al 2007), but three mutants have been identified where the rate of desensitization is slowed (Nayeem et al 2013). For two of these, sidechain rearrangements can explain the phenotype, but for one mutant (GluK2 R775A), the loss of chloride binding was the only apparent structural change.…”

“…For two of these, sidechain rearrangements can explain the phenotype, but for one mutant (GluK2 R775A), the loss of chloride binding was the only apparent structural change. One possible explanation is that the charge balance in the WT receptor tends to destabilize the interface, and the loss of two basic sidechains counteracts this (Nayeem et al 2013).…”

“…), but three mutants have been identified where the rate of desensitization is slowed (Nayeem et al . ). For two of these, sidechain rearrangements can explain the phenotype, but for one mutant (GluK2 R775A), the loss of chloride binding was the only apparent structural change.…”

“…One possible explanation is that the charge balance in the WT receptor tends to destabilize the interface, and the loss of two basic sidechains counteracts this (Nayeem et al . ).…”

Molecular Dynamics Investigation of gluazo, a Photo-Switchable Ligand for the Glutamate Receptor GluK2

“…The LBD features a binding site for ligands, which can be agonists or antagonists. A number of GluK2 LBDs with bound ligands have been resolved by X-ray crystallography [ 10 , 11 ], providing insight into the movement of the receptor and the selectivity of ligand in the receptor gating [ 12 , 13 ].…”