A charge-inverting mutation in the linker region of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors alters agonist binding and gating kinetics independently of allosteric modulators [Molecular Biophysics]

February 18th, 2014 by Harms, J. E., Benveniste, M., Kessler, M., Stone-Roy, L. M., Arai, A. C., Partin, K. M.

AMPA receptors are gated through binding of glutamate to a solvent accessible ligand-binding domain (LBD). Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the LBD to stabilize specific conformations and prevent desensitization. Yelshansky et al. (2004) described a model of an electrostatic interaction between the ligand binding domain and linker region to the pore that regulated channel desensitization. We have conducted a series of experiments to test this hypothesis focusing on the R628E mutation. Using ultra-fast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently-transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500 ms) glutamate application. These changes in receptor kinetics occur through an independent pathway to that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed-cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.
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