2026 Theses Doctoral

Mechanism of gating by AMPA receptors and their auxiliary subunits

Yen, Laura Y.

Excitatory neurotransmission in the central nervous system (CNS) is mediated by ionotropic glutamate receptors (iGluRs), a family of tetrameric, ligand-gated cation-permeable channels. iGluRs are divided into four subclasses: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), kainate receptors (KARs), N-methyl-D-aspartate receptors (NMDARs), and delta (ẟ or GluD) receptors. Throughout the CNS, AMPARs mediate the fast component of excitatory signaling. Within milliseconds of the neurotransmitter glutamate (Glu) binding, AMPARs open their ion channels to permit Na⁺ and K⁺ flux, depolarizing the post-synaptic membrane and triggering downstream signaling. AMPARs are essential for learning, memory, and cognition, and their dysregulation contributes to numerous neurological and psychiatric disorders, including fragile X syndrome, schizophrenia, Parkinson’s disease, and Alzheimer’s disease, underscoring the need for mechanistic and structural insight to inform therapeutic intervention.

Nearly all AMPARs bind to auxiliary subunits—transmembrane proteins that govern receptor assembly, trafficking, pharmacology, and gating kinetics. Among the auxiliary subunits, the transmembrane AMPAR regulatory proteins (TARPs) and cornichon homologs (CNIHs) are the most abundant and well-characterized. Aberrancies in AMPAR–auxiliary subunit interactions contribute to diverse neuropathologies, yet the structural mechanisms underlying their regulation is not completely understood. While cryo-EM structures of homomeric GluA2 receptors and their complexes with type I TARPs (γ2 and γ8) have been extensively characterized, the molecular architecture and gating mechanisms of heteromeric AMPARs—the predominant species in the mammalian brain—and regulation by type II TARPs (γ5 and γ7) remain incomplete, largely due to challenges in co-expression, purification, and conformational heterogeneity.

To address this gap, we used single-particle cryo-electron microscopy (cryo-EM), whole-cell patch-clamp electrophysiology, and molecular-dynamics (MD) simulations to study structure and function of heteromeric GluA1/A2 receptors. We determined GluA1/A2 core structures in the closed, open and desensitized states and analyzed auxiliary subunit contribution to AMPAR architecture and gating. Structural transition from the closed to open state is triggered by glutamate-induced ligand-binding domain (LBD) clamshell closure, inducing tension in the M3–S2 linkers to dilate the channel gate. Auxiliary subunits such as CNIH2 fine-tune this process by stabilizing the open conformation and widening the channel pore without altering LBD clamshell closure. We also find that local asymmetry in the LBD dimer pair correlates with slower recovery from desensitization, establishing LBD dimer symmetry as a key determinant of AMPAR gating kinetics, with auxiliary proteins regulating transitions between functional states.

Building on these findings, we studied modulation of AMPARs by type II TARPs (γ5 and γ7) and CNIH. We characterized the function of GluA2-γ5-CNIH2 and solved its structure in its inactive and desensitized states. We find that γ5 strengthens and prolongs desensitization, whereas CNIH2 weakens desensitization and modulates gating through distinct allosteric effects on the pore and LBDs. CNIH2 also alters ion permeation and pharmacology by stabilizing polyamine binding in the closed state while reducing open-state block and diminishing PMP inhibition. These findings reveal complementary mechanisms by which γ5 and CNIH2 fine-tune AMPAR gating and inhibition to expand the functional diversity of excitatory signaling.

Next, we solved the cryo-EM structures of the GluA1–γ7–CNIH complex in both resting and activated conformational states, demonstrating that the β1-β2 loop of γ7 engages the D2 lobes of LBDs in GluA1 subunits A/C, nearby the conserved KGK motif, suggesting that the A/C subunit LBDs, in addition to B/D subunit LBD-TMD linkers, contribute to functional modulation. Further, LBD clamshell closure in the resting state of AMPARs is independent of TARP identity, indicating that deactivation is primarily governed by TMD interactions.

Collectively, this work defines how heteromeric assembly and auxiliary subunit diversity produce the rich functional repertoire of AMPARs. It establishes a mechanistic framework linking structural asymmetry to kinetic modulation and resolves the first high-resolution architectures of heteromeric GluA1/A2 and γ5- and γ7-bound AMPAR complexes, expanding our understanding of type II TARP function. These findings shed light on the structural underpinnings of excitatory synaptic plasticity and provide a foundation for rational design of therapeutics targeting AMPAR complexes implicated in epilepsy, ischemia, and neurodegenerative disease.