Role of the chemical interactions of the agonist in controlling alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor activation

Abstract

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system. Structures of the isolated ligand binding domain of this receptor have provided significant insight into the large-scale conformational changes, which when propagated to the channel segments leads to receptor activation. However, to establish the role of specific molecular interactions in controlling fine details such as the magnitude of the functional response, we have used a multiscale approach, where changes at specific moieties of the agonists have been studied by vibrational spectroscopy, while large-scale conformational changes have been studied using fluorescence resonance energy transfer (FRET) investigations. By exploiting the wide range of activations by the agonists, glutamate, kainate, and AMPA, for the wild type and Y450F and L650T mutants of the GluR2 subtype, and by using the multiscale investigation, we show that the strength of the interactions at the alpha-amine group of the agonist with the protein in all but one case tracks the extent of activation. Since the alpha-amine group forms bridging interactions at the cusp of the ligand binding cleft, this appears to be a critical interaction through which the agonist controls the extent of activation of the receptor.

Fig. 1. Electrophysiology for wild-type and Y450F

(A) Whole cell current recordings of wild-type and Y450F expressed in HEK 293 cells resulting from exposure to 10 mM glutamate, 10 mM kainate, and 5 mM AMPA with 100 uM cyclothiazide. (B) The maximum currents resulting from glutamate, AMPA, and kainate normalized to the currents mediated by 10 mM AMPA for the wild-type and Y450F and to 10 mM glutamate for L650T.

Fig. 2. Cleft closure in wild type, Y450F-S1S2, and L650T-S1S2

Distances between T394C and S652C as measured by fluorescence resonance energy transfer for the wild type (open circles), S1S2-Y450F mutant (filled squares), and S1S2-L650T mutant (open triangles) plotted as a function of extent of activation.

Fig. 3. Changes at the α-carboxylate and secondary structural modes

Difference FTIR spectra in the region of 1560 cm⁻¹ to 1740 cm⁻¹ between agonist-bound wild type S1S2 and unligated S1S2, agonist-bound S1S2-Y450F and unligated S1S2-Y450F, and agonist bound-L650T and unligated S1S2-L650T for the three agonists AMPA, glutamate, and kainate.

Fig. 4. Changes at the Cys 425 SH stretching mode

Difference FTIR spectra in the region of 2500 cm⁻¹ to 2600 cm⁻¹ between agonist-bound wild type S1S2 and apo S1S2, agonist bound S1S2-Y450F and apo S1S2-Y450F, and agonist bound S1S2-L650T and apo S1S2-L650T for the three agonists AMPA, glutamate, and kainate.