Role of conformational dynamics in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor partial agonism

Abstract

We have investigated the range of cleft closure conformational states that the agonist-binding domains of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors occupy when bound to a series of willardiine derivatives using single-molecule FRET. These studies show that the agonist-binding domain exhibits varying degrees of dynamics when bound to the different willardiines with differing efficacies. The chlorowillardiine- and nitrowillardiine-bound form of the agonist-binding domain probes a narrower range of cleft closure states relative to the iodowillardiine bound form of the protein, with the antagonist (αS)-α-amino-3-[(4-carboxyphenyl)methyl]-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid (UBP-282)-bound form exhibiting the widest range of cleft closure states. Additionally, the average cleft closure follows the order UBP-282 > iodowillardiine > chlorowillardiine > nitrowillardiine-bound forms of agonist-binding domain. These single-molecule FRET data, along with our previously reported data for the glutamate-bound forms of wild type and T686S mutant proteins, show that the mean currents under nondesensitizing conditions can be directly correlated to the fraction of the agonist-binding domains in the "closed" cleft conformation. These results indicate that channel opening in the AMPA receptors is controlled by both the ability of the agonist to induce cleft closure and the dynamics of the agonist-binding domain when bound to the agonist.

FIGURE 1.

a, sample traces of electrophysiological responses evoked by glutamate (Glu), chlorowilladiine (Chloro), nitrowillardiine (Nitro), iodowillardiine (Iodo), and UBP-282 (UBP) under nondesensitizing conditions from cells expressing the T394C/S652C GluA2. Saturating concentrations of Glu (10 mm), chlorowilladiine (1 mm), nitrowillardiine (1 mm), iodowillardiine (1 mm), and UBP-282 (2 mm) were used in the continual presence of 100 μm cyclothiazide. b, summary data showing the electrophysiological response of each willardiine compared with glutamate in the same cell.

FIGURE 2.

a, representative smFRET trace measured from a single agonist-binding domain of GluA2 subunit in complex with chlorowillardiine showing the raw trajectory for donor (blue) and acceptor (red) photons as a function of time. b, the resulting calculated FRET trajectory (green) and its denoised counterpart (black). c and d, histograms of calculated smFRET values from the raw data (c) and following denoising (d).

FIGURE 3.

Denoised ensemble FRET histograms for single agonist-binding domains of GluA2 subunit of AMPA receptors in complex with chlorowillardiine (a), nitrowillardiine (b), iodowillardiine (c), and UBP-282 (d).

FIGURE 4.

a, plot of average single-molecule FRET efficiency over time during a wash from glutamate to glutamate plus iodowillardiine to iodowillardiine alone. Note the progressive decrease in FRET efficiency as glutamate is displaced by iodowillardiine (IW). b, single-molecule histograms from the experiment in a.

FIGURE 5.

Plot of the fraction of the agonist-binding domain of the GluA2 subunit exhibiting FRET efficiencies higher than 0.76 versus normalized mean currents obtained with the corresponding agonists activating the full-length receptors. The data for the glutamate bound and apo form of wild type, as well as glutamate-bound form of T686S mutant, were obtained from Landes et al. (30). ClW, chlorowilladiine; NO₂W, nitrowillardiine; IW, iodowillardiine.