Mechanism of AMPA Receptor Gating Re-Shaped by Auxiliary Proteins

Abstract

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitation in the mammalian central nervous system. Amongst iGluRs, the AMPA receptor (AMPAR) subfamily facilitates the initial depolarization of postsynaptic terminals by gating on a millisecond time scale in response to neurotransmitter binding. In recent years, intact structures of the GluA2 AMPAR determined in several conformational states have been used to develop rudimentary structural models of receptor activation and desensitization. However, it is unclear how such models extrapolate to native AMPARs, which are typically associated with “auxiliary” subunits from one or more protein families. Although auxiliary proteins are known to regulate AMPAR plasma membrane expression and function, the sites at which they functionally interact with receptor subunits have yet to identified and/or characterized. Because we previously demonstrated that the apex of the extracellular ligand-binding domain (LBD) dimer interface is critical for iGluR activation, we investigated whether this site similarly regulates AMPAR-auxiliary protein complexes. GluA2 AMPARs were expressed alone or with auxiliary proteins, and patch-clamp electrophysiology was utilized to assess the functional properties of receptors. Our data reveal that an electrostatic network at the apex of the AMPAR LBD is critical for channel activation, since truncation of residues in this network to alanines generated largely nonfunctional receptors. Interestingly, co-expression of these receptors with auxiliary proteins restored channel gating, indicative that gating was, in part, mediated by interactions outside the LBD dimer interface. Through additional mutagenesis we identified a site, distant from the dimer interface, through which the transmembrane AMPA receptor regulatory protein (TARP) class of auxiliary proteins regulates AMPAR gating, but not permeation properties. In conclusion, our results suggest that the activation of native AMPAR complexes is coordinated by interactions within pore-forming subunits, as well as distinct interactions with auxiliary subunits.