Characterization of a “Hotspot” in the AMPA Receptor Activation Pathway

Ionotropic glutamate receptors (iGluRs) facilitate the bulk of synaptic excitation in the mammalian central nervous system. Structures of full-length, AMPA-type iGluRs (AMPARs) have recently been reported in conformations thought to represent resting, pre-open, and desensitized states. However, it is uncertain what molecular interactions determine whether an agonist-bound AMPAR will favor channel opening or desensitization. We previously described how the activation of kainate-type iGluRs (KARs) is dependent upon occupancy, by sodium, of an electronegative pocket in the ligand-binding domain (LBD). Subsequently, we asked to what extent this pocket, conserved amongst iGluR subfamilies, regulates AMPAR activation. To investigate this subject we utilized electrophysiological (outside-out patch) recordings from iGluR subunits transiently expressed in HEK 293 cells, as well as molecular dynamics (MD) simulations. Unlike the KAR subunit GluK2, receptors comprised of the AMPAR subunit GluA2 did not require occupancy of the pocket by a positive charge to activate. Interestingly, a lithium ion has been detected in the pocket of recent crystal structures of the GluA2 LBD. The effect of lithium in the external recording solution was to dramatically slow the desensitization kinetics of GluA2. MD simulations supported an increased affinity of the site for lithium versus sodium, and predicted that lithium binding holds subunits closer together. Through disrupting an inter-subunit electrostatic bridge adjacent to the “cation” pocket, the effect of lithium was greatly attenuated. In fact, the removal of key charges at this interface produced receptors barely capable of activation, although the functional deficit was rescued by the modulator cyclothiazide or co-expression with auxiliary subunits. We propose that when electrostatic interactions at the apex of the LBD are stabilized, AMPARs are primed for activation, whereas the disruption of these interactions directs receptors to desensitized states upon agonist binding.