Using positive allosteric modulators to determine mechanisms of GluR2 desensitization and deactivation

Abstract

Recent crystallographic and functional analyses of AMPA receptors have identified a common binding pocket for two classes of positive allosteric AMPA receptor modulators, cyclothiazide (CTZ) and AMPAkine CX614. Based on differences in modulator binding and function, those experiments have provided a working model consisting of two distinct pathways for channel desensitization and deactivation where rearrangement of the ligand binding core at the interdimer interface is responsible for receptor desensitization, and cleft opening of the ligand binding core is responsible for deactivation. This study focuses on comparing structural and functional data for GluR2 flip and flop in the presence of four AMPA receptor modulators: CTZ, CX614, LY506091, and LY2152080. Additionally we have tested the effects of point mutations at the three subdomains implicated in receptor deactivation (Hinge 1 and Hinge 2) and desensitization (flip/flop) and modulation. We have also used computer simulations to model the effects of slowing cleft-opening, entry into desensitization and channel closure rate on macroscopic deactivation and desensitization kinetics. Together, these data test and refine the current model of AMPA receptor gating and modulation that has been based on static snapshots of crystal structures. These findings lend support to the idea that deactivation and desensitization (as well as the modulation of each) are, in fact, inextricably coupled, likely with only one mechanism for closing the receptor gate.