Conformational Flexibility of the Ligand-Binding Domain Dimer in Kainate Receptor Gating and Desensitization

Abstract

AMPA-and kainate (KA)-selective ionotropic glutamate receptors (iGluRs) respond to agonist by opening (gating), then closing (desensitizing) in quick succession. Gating has been linked to agonist-induced changes within the ligand-binding domain (LBD), and desensitization to rearrangement of a dimer formed by neighboring LBDs. To explore the role of dimer conformation in both gating and desensitization, we compared the conformational effects of two kainate receptor mutants. The first, GluK2-D776K, blocks desensitization of macroscopic current responses ("macroscopic desensitization"). The second, GluK2-M770K, accelerates macroscopic desensitization and eliminates the effects of external ions on channel kinetics. Using structures determined by x-ray crystallography, we found that in both mutants the introduced lysines act as tethered cations, displacing sodium ions from their binding sites within the dimer interface. This results in new inter-and intra-protomer contacts in D776K and M770K respectively, explaining the effects of these mutations on dimer stability and desensitization kinetics. Further, chloride binding was unaffected by the M770K mutation, even though binding of sodium ions has been proposed to promote dimer stability by stabilizing anion binding. This suggests sodium binding may affect receptor function more directly than currently supposed. Notably, we also observed a ligand-specific shift in dimer conformation when comparing LBD dimers in complex with glutamate or the partial agonist KA, revealing a previously unidentified role for dimer orientation in iGluR gating.

Binding site and interlobe interactions of the ionotropic glutamate receptor GluK3 ligand binding domain revealed by high resolution crystal structure in complex with (S)-glutamate

“…Two such GluA2-like dimeric structures of GluK1 (Mayer et al, 2006;Unno et al, 2010) and also two of GluK2 (Chaudhry et al, 2009;Nayeem et al, 2011) have been determined in complex with glutamate. However, in other structures with glutamate either monomers (Mayer, 2005;Unno et al, 2010) or a different dimer (Mayer, 2005;Naur et al, 2005) were seen.…”

“…It is also possible that the dimer observed is merely a consequence of crystal packing. However, it is known that introducing mutations in dimer interfaces might lead to significant and unexpected effects in receptor function (Nayeem et al, 2011), so a functional role of the observed dimer versus crystal packing effects cannot be completely ruled out.…”

Functional Insights from Glutamate Receptor Ion Channel Structures

“…No similar drugs have been reported for kainate receptors, although their disclosure likely remains only a matter of time. However, crystal structures for LBD dimer assemblies of GluK1, GluK2 and GluD2 reveal novel binding sites for Na + , Ca 2+ and Cl − ions (84, 120–123), which act as counter charges at polar sites on the dimer interface, thereby increasing LBD dimer stability and reducing desensitization (124). …”

Correlating efficacy and desensitization with GluK2 ligand-binding domain movements

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Figure 1.Apical interactions within GluK2 LBD dimer. ( a ) The GluK2 WT LBD dimer (PDB code 2 xxr ) [7] showing ligand (black), domains D1 and D2, the twofold axis and the Na + and Cl − ions (purple and green, respectively). Key D1:D1 interactions are shown inset on protomer B; pink surface from data in reference [8] and grey surface from this study.

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