Molecular determinants of AMPA receptor subunit assembly

AMPA-type (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) glutamate receptors (AMPARs) mediate post-synaptic depolarization and fast excitatory transmission in the central nervous system. AMPARs are tetrameric ion channels that assemble in the endoplasmic reticulum (ER) in a poorly understood process. The subunit composition determines channel conductance properties and gating kinetics, and also regulates vesicular traffic to and from synaptic sites, and is thus critical for synaptic function and plasticity. The distribution of functionally different AMPARs varies within and between neuronal circuits, and even within individual neurons. In addition, synapses employ channels with specific subunit stoichiometries, depending on the type of input and the frequency of stimulation. Taken together, it appears that assembly is not simply a stochastic process. Recently, progress has been made in understanding the molecular mechanisms underlying subunit assembly and receptor biogenesis in the ER. These processes ultimately determine the size and shape of the postsynaptic response, and are the subject of this review.

Introduction

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic transmission in vertebrate central nervous systems (CNSs) [1]. Three subfamilies of glutamate-gated, cation-selective channels mediate rapid signalling: AMPA, NMDA (N-methyl-d-aspartate) and kainate receptors. They play pivotal roles in synapse formation and maintenance, and in various forms of synaptic plasticity 2, 3. In addition, dysfunction of these receptors results in diverse acute and chronic neurological disorders. iGluRs are widely distributed in the CNS, where they fulfil different functions. AMPA-type glutamate receptors (AMPARs) mediate primary depolarization in glutamatergic transmission, and their exceptionally fast kinetics conveys ‘point-to-point’ signalling 2, 4.

In contrast to the skeletal nicotinic acetylcholine receptor, which is built in a predetermined fashion [5], AMPARs are expressed, like many other ion channels, in various subunit combinations; they assemble from four subunits, GluR1–4 (or GluRA–D), into ion channel tetramers 1, 6, 7, 8, 9. The selectivity of the assembly process, which determines channel functional properties, is poorly understood. The subunit stoichiometry also regulates vesicular traffic 1, 10, 11 and synapse-specific targeting of channel tetramers 12, 13, 14, and thereby directly impacts synaptic efficacy and plasticity. Moreover, accumulating evidence suggests that the AMPAR composition is not static, but can be altered in response to certain inputs 15, 16, 17, 18, 19, 20, 21.

In analogy to K⁺ channels [22], AMPARs are thought to assemble as dimers of dimers 9, 23, 24. The N-terminal domain mainly mediates dimer formation; subsequent tetramerisation involves the extracellular S2 loop and the transmembrane segments (including the pore loop; Figure 1a) [23]. Receptor assembly occurs at the endoplasmic reticulum (ER) membrane. Exit from the ER is under stringent quality control, which monitors correct subunit folding and assembly [25]. The efficiency of these processes impacts on ER export kinetics and thereby determines the number of channels available for expression at synapses; this will ultimately tune the responsiveness of a neuron. Interestingly, recent studies indicate that conformational alterations associated with gating motions, such as ligand binding and desensitization (i.e. channel closure in ligand bound state), take place in the ER lumen, where they are sensed by the quality control machinery 26, 27, 28, 29, 30, 31. These findings add iGluRs to a growing list of ER client proteins that require ligands or ‘chemical chaperones’ for efficient folding and export from the ER [32].

In addition to the subunit stoichiometry, AMPAR function is diversified further by RNA processing events, including alternative splicing and RNA editing [1]. Splicing of mutually exclusive exons (termed flip/flop) within the ligand-binding domain (LBD) modulates desensitization kinetics [33]. These two alternative exons are present in all four subunits and are common to vertebrate AMPARs (Figure 2) [34]. Also, RNA editing at two sites modulates AMPAR function — the R/G site within the LBD of GluR2–4 and the Q/R site in the pore loop of GluR2 (Figure 1a; Box 1) 35, 36. These sites are located within subunit interfaces (Figure 1b) and intimately impact receptor assembly 30, 37. Below, we discuss the role of individual domains in the assembly process and review structural parameters of their interaction surfaces. We then describe how these interfaces are modulated by RNA editing and how these modifications evolved in the iGluR lineage (Figure 2; Box 2). Finally, we consider how gating motions, initiated by ligand binding in the ER, contribute to receptor folding and assembly.