Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

摘要

class="head no_bottom_margin" id="sec1title">IntroductionAMPA-type glutamate receptors (AMPARs) mediate fast excitatory transmission and are crucial for various forms of synaptic plasticity (). Their varied kinetic behavior (), as well as their calcium permeability and voltage-dependent block by polyamines (), varies between brain regions and appear to be adapted to the specific function of a given circuit (). These properties depend on the nature and mRNA processing status of the four pore-forming subunits (GluA1–GluA4) () and on the type and stoichiometry of AMPAR auxiliary subunits ().Four families of auxiliary subunits have been identified: transmembrane AMPAR regulatory proteins (TARPs) (), cornichons (), CKAMP44 (), and GSG1L (). Most of these alter AMPAR gating and confer effects that can be specific for a given synapse or cell. TARPs were the first identified bona fide AMPAR auxiliary proteins, modifying both AMPAR function and trafficking. Based on their modulatory actions, TARPs have been classified as type 1a (γ-2 and γ-3), type 1b (γ-4 and γ-8), and type 2 (γ-5 and γ-7) (). TARP-like modulation of AMPARs has also been seen in invertebrates () and thus appears highly conserved.The precise nature of the AMPAR/TARP interaction and thus the mechanism underlying gating modulation are poorly understood. Both the AMPAR transmembrane region and the ligand binding domain (LBD) have been implicated in TARP interactions responsible for the modulation of ligand efficacy, pharmacology, gating, and pore properties (). Experiments using domain swapping between subtypes have identified TARP regions that are involved in regulating AMPARs. These include the extracellular loop (Ex1), the transmembrane sector, and the C terminus. Specifically, the TARP C tail appears critical for receptor trafficking and mediation of kinetic effects, while Ex1 influences both the efficacy of the partial agonist kainate and AMPAR kinetics ().The most distal AMPAR domain, the N-terminal domain (NTD), is expected to be beyond the “reach” of the associated TARP. Apart from a role in subunit assembly, no clear function has been ascribed to this large and most sequence-diverse domain (href="#bib17" rid="bib17 bib28" class=" bibr popnode">Hansen et al., 2010; Kumar and Mayer, 2013), although deletion of the NTD slows desensitization kinetics (href="#bib3" rid="bib3 bib36 bib41" class=" bibr popnode">Bedoukian et al., 2006; Möykkynen et al., 2014; Pasternack et al., 2002). In stark contrast, the NTD of the N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR) mediates allosteric regulation of channel open probability (href="#bib40" rid="bib40" class=" bibr popnode">Paoletti, 2011) in a subunit-specific manner, rendering the NTD an important target for selective NMDAR drugs (href="#bib32" rid="bib32" class=" bibr popnode">Mony et al., 2009). NTD-mediated allostery in NMDARs has been shown to involve the ∼16-residue peptide linkers that connect the NTD to the LBD (href="#bib13" rid="bib13 bib33 bib68" class=" bibr popnode">Gielen et al., 2009; Mony et al., 2011; Yuan et al., 2009).Here we show that the AMPAR NTD plays a previously unrecognized role in signaling. Shortening of the NTD-LBD linkers altered desensitization rates and recovery from the desensitized state and increased the steady-state response. These gating effects were TARP dependent and TARP specific. Using peptide arrays, we mapped the GluA2/TARP contact region and identified TARP binding sites on the NTD. On the LBD, TARP contact points mapped to functionally critical sites, including the ligand binding cleft, the flip/flop region, and the linkers that connect the LBD to the ion channel. We also determined the sites on the TARP that are contacted by the AMPAR and assessed their functional role using corresponding TARP mutants. Our results provide detailed insights into the molecular interactions of TARPs with AMPARs and show that these include the distal NTD. This subunit-specific TARP regulatory site may permit fine tuning of AMPAR signaling and provide a target for subunit-selective drugs.