Chemical Interplay in the Mechanism of Partial Agonist Activation in α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors

Abstract

Abstractα-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, one subtype in the family of ionotropic glutamate receptors, are the main receptors responsible for excitatory signaling in the mammalian central nervous system. Previous studies utilitizing the isolated ligand binding domain of these receptors have provided insight into the role of specific ligand-protein interactions in mediating receptor activation. However, these studies relied heavily on the partial agonist kainate, in which the α-amine group is constrained in a pyrrolidine ring. Here we have studied a series of substituted and unsubstituted willardiines with primary α-amine groups similar to that of the full agonist glutamate whose activation can be varied depending on the size of the substituent. The specific ligand-protein interactions in the mechanism of partial agonism in this subtype were investigated using vibrational spectroscopy and the large scale conformational changes in the ligand binding domain were studied with fluorescence resonance energy transfer (FRET). These investigations show that the strength of the interaction at the α-amine group correlates with the extent of cleft closure and extent of activation, with the agonist of higher efficacy showing larger cleft closure and stronger interactions at this group, suggesting that this is one of the mechanisms by which the agonist controls receptor activation.α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, one subtype of ionotropic glutamate receptors, are responsible for mediating the majority of excitatory synaptic signaling in the central nervous system. Activation of these channels occurs when an agonist, such as glutamate, binds in the bilobed extracellular ligand binding domain of the receptor, initiating a series of conformational changes that lead to the formation of cation selective channels (1-4).Recent vibrational spectroscopic investigations (5-8) in combination with fluorescence resonance energy transfer (FRET) studies (5,8,9) on the isolated ligand binding domain of the GluR2 subunit (GluR2-S1S2) have provided insight into the role of specific ligand-protein interactions in mediating AMPA receptor activation. By using these techniques to study activation by the three ligands kainate, AMPA, and glutamate on wild type and mutant proteins, which provide a wide spectrum of activations, a correlation was developed between the changes at specific ligand-protein interactions and extent of activation of the channel. These studies suggest that the strength of the interaction at the α-amine group correlates in most cases to the § Address correspondence to: Vasanthi Jayaraman, Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, 6431 Fannin St., Houston, Texas, 77030; (5,6). However, it should be noted that these correlations depended heavily on conclusions from the partial agonist kainate, which is inherently different in structure compared to glutamate and AMPA because it...

NMR Spectroscopy of the Ligand-Binding Core of Ionotropic Glutamate Receptor 2 Bound to 5-Substituted Willardiine Partial Agonists

“…Interestingly, Fourier transform infrared resonance spectroscopy measurements at pH 7.4 suggest that the bound form is protonated based on difference spectra for the ligand carbonyl vibrations (V. Jayaraman, personal communication). 62,63 If this is the case for all the ligands examined in the present study, then the R ex and Î data may indicate that the willardiines bind stably to sites in lobe 1 on the NMR timescale but give rise to significant μs-ms timescale dynamics at the ligandlobe 2 interface for the less potent forms.…”

Role of Conformational Dynamics in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Partial Agonism

“…However, there were several structures such as those of the glutamate-bound form of the T686A mutant and the structures of the AMPA bound form of the L650T mutant that do not follow this trend (22,(25)(26)(27). Ensemble FRET investigations with the wild type and L650T mutant were consistent with the x-ray structures, further validating these deviations from the cleft closure hypothesis (28,29).…”

Mechanism of Partial Agonism at the GluR2 AMPA Receptor: Measurements of Lobe Orientation in Solution

“…The efficacies are taken from Jin et al (5) and Mankiewicz et al (12) and are relative to the full agonist, glutamate. When a given agonist was tested in both studies, the average value was used and the standard error of the mean shown as the error bar.…”

“…In addition to the crystal structures, the internal dynamics of each lobe have been studied with NMR spectroscopy (8–11) and the relative strengths of hydrogen bonds have been accessed with FTIR (12–14) and NMR (9) studies. The overall picture is that Lobe 1 (Figure 1) is relatively rigid on the μs-ms timescale, with Lobe 2 exhibiting characteristic internal motions that vary between full and partial agonists (8–11).…”