Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that form transmembrane, cation-permeable channels. iGluRs exist as three distinct subfamilies according to their agonist specificity and amino acid sequence: α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), kainate receptors, and N
-methyl-D-aspartate receptors (NMDARs). Activation of iGluRs is critical for induction of some forms of long-term potentiation (LTP), a type of synaptic plasticity associated with learning and memory [1
]. iGluRs play crucial roles in normal neuronal function and in neurological disease, and thus are being actively pursued as therapeutic targets for the treatment of amyotrophic lateral sclerosis, neuropathic pain, major depression, Alzheimer’s disease, and Parkinson’s disease.
Despite having divergent functional properties, iGluR family members adopt a common modular architecture. A typical iGluR subunit contains four distinct domains: an extracellular amino-terminal domain (~ 400 residues, ATD), an extracellular ligand-binding domain (~ 300 residues, LBD), three transmembrane domains (TM1-3) plus a re-entrant pore loop (P), and an intracellular carboxy-terminal domain (). Active iGluR channels are tetramers formed exclusively by assembly of subunits within the same subfamily. This process is thought to be mediated in part by the ATD through a mechanism that is not fully understood.
Historically, structural biology has made significant contributions to our understanding of iGluR function. The first crystal structure of an iGluR, the LBD of GluA2, was determined in 1998 [3
]. Since then, more than a hundred high-resolution crystal structures of iGluR LBD have been reported, including subunits from all three subfamilies of iGluR either in the apo form or in complex with a variety of agonists, antagonists, and modulators. Collectively, these structures have established the detailed molecular mechanisms underlying iGluR channel activation, inhibition, and desensitization [4
]. Nevertheless, the first crystal structure of a full length iGluR ion channel, the homomeric GluA2, was not accomplished until 2009 [6
]. This extraordinary advance has yielded unprecedented information on the molecular architecture and symmetry of iGluR. Structural studies on full-length iGluRs are exceptionally difficult to perform, and to date have yielded only a single snapshot of a homomeric GluA2 bound with an antagonist. It is therefore not surprising that our understanding of iGluR structure and function continues to be derived from studies with isolated recombinant ATDs and LBDs.
In contrast to the well-characterized LBD, much less is known about the molecular properties of the ATD, even though this domain is crucial for the physiological function of iGluRs. For example, the spontaneous mouse mutation hotfoot
is a recessive mutation characterized by cerebellar ataxia and jerky movement of the hind limbs, and is often caused by in-frame deletions of various regions of the ATD of GluRδ2 [7
]. The functional activities of ATD fall into three general categories. (i) It is widely accepted that the iGluR-ATD guides receptor subfamily-specific assembly, ensuring that only subunits within the same subfamily assemble with one another [8
]. Interestingly, the subfamily-specific assembly of tetrameric voltage-gated Shaker K+
channels is also determined by the amino-terminal T1 domain [12
]. (ii) The NMDAR-ATD modulates receptor function by providing binding sites for allosteric modulators that include protons, zinc ions, polyamines, and small organic molecules such as ifenprodil [1
]. However, no small molecules have been shown to bind the AMPAR-ATD or the ATD of kainate receptors. (iii) The ATD resides within the synaptic cleft, and thus may be involved in protein-protein interactions. By doing so, the ATD could regulate both dendritic spine morphogenesis and presynaptic stability [17
]. Some ATD binding partners have been identified, including neuronal pentraxins (Narp, NP1, and NPR) [19
] and N-cadherin [21
] that bind to AMPAR-ATD, and an ephrin receptor that binds to NMDAR [22
Our limited knowledge of the structure and function of the ATD is in part due to the challenge associated with recombinant protein production. Large-scale protein expression of the ATD using insect and mammalian cells has been successful only recently, and has resulted in a number of ATD structures, including the ATDs of GluA2, GluK2, GluK3, GluK5, and GluN2B [23
]. Structural analyses have revealed several unique features of ATDs within each subfamily of iGluRs with respect to the protomer structure, subfamily-specific subunit assembly, and competence for ligand binding. These findings have clarified how the ATD prevents subunits from different subfamilies from assembling into a tetramer. However, the role played by the ATD in driving assembly of different subunits within a subfamily remains unclear. This is a critical process, as most native AMPAR channels are heteromers that co-assemble with the GluA2 subunit, which serves to decrease channel permeability to calcium ions. This problem has motivated us to perform structural studies on other subunits of AMPARs to complement the structure of the GluA2-ATD, which we reported in 2009 [23
]. Here, we report the structure of the GluA1-ATD at 2.5 Å resolution. The crystal structures of GluA3 and GluN1-ATD were reported while this manuscript was in preparation [29