Chemical interplay in the mechanism of partial agonist activation in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors

Abstract

Alpha-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 alpha-amine group is constrained in a pyrrolidine ring. Here we have studied a series of substituted and unsubstituted willardiines with primary alpha-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 alpha-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.

Figure 1

Structures of AMPA receptor full agonists glutamate and AMPA and partial agonists kainate and 5-‘R’-willardiine.

Figure 2. Activation profile of the series of willardiines

Maximum currents elicited under non-desensitizing conditions by saturating concentrations of glutamate (10 mM), ClW (1mM), FW (1mM), IW (1mM), and HW (1 mM), normalized to maximum response mediated by glutamate.

Figure 3. FRET to measure the extent of cleft closure with various agonists

Extent of activation (normalized to glutamate activation) versus distances between residues 394 and 652 in S1S2-T394C-S652C as determined from the fluorescence lifetimes for the series of willardiines and agonists glutamate, AMPA, and kainate (9).

Figure 4. Environment of willardiines free in solution

Difference FTIR spectra in the region of 1580–1740 cm⁻¹ between FW and buffer, HW and buffer, IW and buffer, and ClW and buffer.

Figure 5. Changes in interaction at α-carboxylate and protein secondary structure

Difference FTIR spectra in region of 1580 cm⁻¹–1740 cm⁻¹ for FW-bound GluR2-S1S2 and apo GluR2-S1S2, HW-bound GluR2-S1S2 and apo GluR2-S1S2, IW-bound GluR2-S1S2 and apo GluR2-S1S2, and ClW-bound GluR2-S1S2 and apo GluR2-S1S2.

Figure 6. Interaction at the α-amine group as determined by the Cys 425 S-H stretching mode

Difference FTIR spectra in region of 2500–2600 cm⁻¹ between apo GluR2-S1S2 and buffer, IW-bound GluR2-S1S2 and buffer, ClW-bound GluR2-S1S2 and buffer, FW-bound GluR2-S1S2 and buffer, and HW-bound GluR2-S1S2 and buffer.

Figure 7. Relationship between strength of interaction at α-amine group and extent of activation

Plot of S-H stretching frequency vs. extent of activation for apo and agonist-bound GluR2-S1S2 (6, 9), illustrating the correlation between the strength of interaction at α-amine group and the extent of activation.