Differential regulation of tetramerization of the AMPA receptor glutamate-gated ion channel by auxiliary subunits.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) auxiliary subunits are specialized, nontransient binding partners of AMPARs that modulate AMPAR channel gating properties and pharmacology, as well as their biogenesis and trafficking. The most well-characterized families of auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs), cornichon homologs (CNIHs), and the more recently discovered GSG1-L. These auxiliary subunits can promote or reduce surface expression of AMPARs (composed of GluA1-4 subunits) in neurons, thereby impacting their functional role in membrane signaling. Here, we show that CNIH-2 enhances the tetramerization of WT and mutant AMPARs, presumably by increasing the overall stability of the tetrameric complex, an effect that is mainly mediated by interactions with the transmembrane domain of the receptor. We also find CNIH-2 and CNIH-3 show receptor subunit-specific actions in this regard with CNIH-2 enhancing both GluA1 and GluA2 tetramerization, whereas CNIH-3 only weakly enhances GluA1 tetramerization. These results are consistent with the proposed role of CNIHs as endoplasmic reticulum cargo transporters for AMPARs. In contrast, TARP γ-2, TARP γ-8, and GSG1-L have no or negligible effect on AMPAR tetramerization. On the other hand, TARP γ-2 can enhance receptor tetramerization but only when directly fused with the receptor at a maximal stoichiometry. Notably, surface expression of functional AMPARs was enhanced by CNIH-2 to a greater extent than TARP γ-2, suggesting that this distinction aids in maturation and membrane expression. These experiments define a functional distinction between CNIHs and other auxiliary subunits in the regulation of AMPAR biogenesis.

Differential regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) receptor tetramerization by auxiliary subunits.

AMPA receptor (AMPAR) auxiliary subunits are specialized, non-transient binding partners of AMPARs that modulate their ion channel gating properties and pharmacology, as well as their biogenesis and trafficking. The most well characterized families of auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs) and cornichon homologs (CNIHs) and the more recently discovered GSG1-L. These auxiliary subunits can promote or reduce surface expression of AMPARs in neurons, thereby impacting their functional role in membrane signaling. Here, we show that CNIH-2 enhances the tetramerization of wild type and mutant AMPARs, possibly by increasing the overall stability of the tetrameric complex, an effect that is mainly mediated by interactions with the transmembrane domain of the receptor. We also find CNIH-2 and CNIH-3 show receptor subunit-specific actions in this regard with CNIH-2 enhancing both GluA1 and GluA2 tetramerization whereas CNIH-3 only weakly enhances GluA1 tetramerization. These results are consistent with the proposed role of CNIHs as endoplasmic reticulum cargo transporters for AMPARs. In contrast, TARP γ-2, TARP γ-8, and GSG1-L have no or negligible effect on AMPAR tetramerization. On the other hand, TARP γ-2 can enhance receptor tetramerization but only when directly fused with the receptor at a maximal stoichiometry. Notably, surface expression of functional AMPARs was enhanced by CNIH-2 to a greater extent than TARP γ-2 suggesting that this distinction aids in maturation and membrane expression. These experiments define a functional distinction between CNIHs and other auxiliary subunits in the regulation of AMPAR biogenesis.

NMDA Receptors Require Multiple Pre-opening Gating Steps for Efficient Synaptic Activity.

NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate fast excitatory synaptic transmission in the nervous system. Applying glutamate to outside-out patches containing a single NMDAR, we find that agonist-bound receptors transition to the open state via two conformations, an "unconstrained pre-active" state that contributes to fast synaptic events and a "constrained pre-active" state that does not. To define how glutamate drives these conformations, we decoupled the ligand-binding domains from specific transmembrane segments for GluN1 and GluN2A. Displacements of the pore-forming M3 segments define the energy of fast opening. However, to enter the unconstrained conformation and contribute to fast signaling, the GluN2 pre-M1 helix must be displaced before the M3 segments move. This pre-M1 displacement is facilitated by the flexibility of the S2-M4 of GluN1 and GluN2A. Thus, outer structures-pre-M1 and S2-M4-work in concert to remove constraints and prime the channel for rapid opening, facilitating fast synaptic transmission.

Lupus autoantibodies act as positive allosteric modulators at GluN2A-containing NMDA receptors and impair spatial memory.

Patients with Systemic lupus erythematosus (SLE) experience various peripheral and central nervous system manifestations including spatial memory impairment. A subset of autoantibodies (DNRAbs) cross-react with the GluN2A and GluN2B subunits of the NMDA receptor (NMDAR). We find that these DNRAbs act as positive allosteric modulators on NMDARs with GluN2A-containing NMDARs, even those containing a single GluN2A subunit, exhibiting a much greater sensitivity to DNRAbs than those with exclusively GluN2B. Accordingly, GluN2A-specific antagonists provide greater protection from DNRAb-mediated neuronal cell death than GluN2B antagonists. Using transgenic mice to perturb expression of either GluN2A or GluN2B in vivo, we find that DNRAb-mediated disruption of spatial memory characterized by early neuronal cell death and subsequent microglia-dependent pathologies requires GluN2A-containing NMDARs. Our results indicate that GluN2A-specific antagonists or negative allosteric modulators are strong candidates to treat SLE patients with nervous system dysfunction.