Structure and organization of AMPA receptor-TARP complexes in the mammalian cerebellum.

AMPA receptors (AMPARs) are multimodal transducers of glutamatergic signals throughout the brain. Their diversity is exemplified in the cerebellum: At afferent synapses, AMPARs mediate high-frequency excitation, whereas in Bergmann glia (BG) they support calcium transients that modulate synaptic transmission. This spectrum arises from different combinations of core subunits (GluA1-4), auxiliary proteins, and posttranscriptional modifications. Using mass spectrometry, cryo–electron microscopy, and electrophysiology, we characterize major cerebellar AMPARs in pigs: calcium-impermeable GluA2/A4 heteromers with four transmembrane AMPAR regulatory protein (TARP) subunits, mainly neuronal in origin, and BG-specific, calcium-permeable GluA1/A4 heteromers containing two type II TARPs. We also showed that GluA4 receptors frequently exhibit compact N-terminal domains that promote their synaptic delivery. Our study defines the organizational principles of mammalian cerebellar AMPAR complexes and reveals how different receptor subtypes support cell type–specific functions. Editor's summary: Mammalian AMPA receptors are tetrameric glutamate-responsive ion channels localized at synapses throughout the nervous system. Their architecture and the assembly process in their native context remain to be fully elucidated. Scrutton et al. purified GluA4-containing channels from porcine cerebellum, where this core subunit is particularly enriched, and subjected them to quantitative mass spectrometry, cryo–electron microscopy analysis, and electrophysiology. AMPA receptors derived from neurons and glia differed in subunit composition, suggesting that this different composition supports cell type–specific channel functions. —Mattia Maroso INTRODUCTION: Excitatory synaptic transmission in the brain is mediated by ionotropic glutamate receptors—cation channels that open in response to glutamate released from presynaptic terminals. Their signaling is fundamental for higher cognitive processes such as learning and memory. Among them, AMPA receptors (AMPAR) are the most diverse as a result of their combinatorial assembly from four subunits, GluA1-4, which determine channel kinetics and calcium permeability. Further diversification is achieved through the inclusion of auxiliary subunits, such as the six-membered transmembrane AMPAR regulatory proteins (TARP) family. AMPAR diversity is exemplified in the cerebellum, a brain structure dedicated to sensorimotor learning and control. Here, GluA4 subunits are enriched and distributed across both neurons and glial cells to support specialized signaling functions. However, the organization and subunit stoichiometries of these prevalent GluA4-type AMPARs remained unclear. RATIONALE: Our study aimed to characterize the organization of GluA4-containing AMPAR in the mammalian cerebellum. Using a GluA4-specific nanobody, we selectively isolated GluA4 subtypes from pig cerebella and analyzed their molecular composition by mass spectrometry (MS) and cryo–electron microscopy (cryo-EM). This approach revealed two major receptor populations, either calcium-impermeable [(CI); containing GluA2 subunits] or calcium-permeable [(CP); lacking GluA2]. Each group was associated with distinct TARP subtypes. In addition, we performed electrophysiological recordings from cerebellar brain slices and from defined receptor combinations reconstituted into HEK293 cells, to further investigate subunit composition and gating properties. RESULTS: Our MS data reveal that GluA4 AMPARs primarily associate with GluA1 subunits and predominantly harbor TARPs, whereas other auxiliary subunits were present at substantially lower levels. Moreover, these auxiliary subunits are expressed substoichiometrically, suggesting the AMPARs are not fully occupied at their four available binding sites, which has notable implications for receptor gating and synaptic localization. The MS data were mirrored by the cryo-EM structural analysis, which revealed two major GluA4-containing receptor populations: one associated with two TARPs and the other with four. These two groups revealed a further key distinction: The two-TARP "hexameric" receptors largely lacked GluA2 subunits, whereas the "octameric" (four-TARP) receptors incorporated GluA2. Consequently, the GluA1/A4 receptor hexamers are calcium-permeable and originate primarily from glial cells, whereas the mixed GluA2/A4 population is calcium-impermeable and predominantly of neuronal origin. Furthermore, the two classes differ in their associated TARPs, which markedly differ in their functional properties—hexamers associate with type II, whereas octamers contain type I. Electrophysiological recordings revealed that the CP GluA1/4 heteromers largely associate with TARP-γ7. A hallmark feature of GluA4 is its compact, tetrameric N-terminal domain (NTD) tier, which drives synaptic receptor localization. The NTD extends into the synaptic cleft, where it interacts with a dedicated anchoring machinery dependent upon its tetrameric architecture. Rupturing this NTD assembly by targeted mutation substantially reduced synaptic responses, as observed previously for GluA2. CONCLUSION: We demonstrate the various stoichiometries of cerebellar GluA4 AMPARs associated with TARP auxiliary subunits. Our preparation was dominated by CP GluA1/A4 heteromers, containing two TARPs, which are enriched in glial cells, whereas the CI GluA2/A4-containing receptors carry four TARPs and derive from the vastly abundant cerebellar granule neurons. These organizational principles illuminate how different AMPAR subtypes support cell type–specific functions in the cerebellar circuitry. Analysis of GluA4 AMPA receptors from pig cerebellum.: (Top left) Pig brain schematic, with the GluA4-enriched cerebellum shown in yellow. (Top right) Zoom-in to the cerebellar circuitry with granule cells (GCs) harboring GluA2, thus CI and Bergmann glia [(BG) lacking GluA2; CP] are shown. Bottom (left): schematized MS data highlighting the abundance of GluA1 and GluA4 together with TARP auxiliary subunits. (Right): Models of GluA2-containing AMPAR octamer and GluA2-lacking hexamer enriched in GC and BG, respectively. These differ in their electrophysiological characteristics. [ABSTRACT FROM AUTHOR]