Single-molecule FRET experiments monitoring AMPA receptor dynamics reveal the energy landscape that the receptor samples and establish that agonists control the level of activation by modulating the fraction of protein in the closed-cleft conformation. AMPA receptors mediate fast excitatory neurotransmission by converting chemical signals into electrical signals, and thus it is important to understand the relationship between their chemical biology and their function. We used single-molecule fluorescence resonance energy transfer to examine the conformations explored by the agonist-binding domain of the AMPA receptor for wild-type and T686S mutant proteins. Each form of the agonist binding domain showed a dynamic, multistate sequential equilibrium, which could be identified only using wavelet shrinkage, a signal processing technique that removes experimental shot noise. These results illustrate that the extent of activation depends not on a rigid closed cleft but instead on the probability that a given subunit will occupy a closed-cleft conformation, which in turn is determined not only by the lowest energy state but also by the range of states that the protein explores.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligand-binding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the α-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist.

Fast excitatory synaptic signaling in the mammalian brain is mediated by AMPA-type ionotropic glutamate receptors. In neurons, AMPA receptors co-assemble with auxiliary proteins, such as stargazin, which can markedly alter receptor trafficking and gating. Here, we used luminescence resonance energy transfer measurements to map distances between the full-length, functional AMPA receptor and stargazin expressed in HEK293 cells and to determine the ensemble structural changes in the receptor due to stargazin. In addition, we used single-molecule fluorescence resonance energy transfer to study the structural and conformational distribution of the receptor and how this distribution is affected by stargazin. Our nanopositioning data place stargazin below the AMPA receptor ligand-binding domain, where it is well poised to act as a scaffold to facilitate the long-range conformational selection observations seen in single-molecule experiments. These data support a model of stargazin acting to stabilize or select conformational states that favor activation.

Many neurological disorders show an increased prevalence of GluA2-lacking, Ca²⁺-permeable AMPA receptors (CP-AMPARs), which dramatically alters synaptic function. However, the molecular mechanism underlying this distinct synaptic plasticity remains enigmatic. Here, we show that nerve injury potentiates postsynaptic, but not presynaptic, CP-AMPARs in the spinal dorsal horn via α2δ-1. Overexpressing α2δ-1, previously regarded as a Ca²⁺ channel subunit, augments CP-AMPAR levels at the cell surface and synapse. Mechanistically, α2δ-1 physically interacts with both GluA1 and GluA2 via its C terminus, inhibits the GluA1/GluA2 heteromeric assembly, and increases GluA2 retention in the endoplasmic reticulum. Consequently, α2δ-1 diminishes the availability and synaptic expression of GluA1/GluA2 heterotetramers in the spinal cord in neuropathic pain. Inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-AMPAR complex fully restores the intracellular assembly and synaptic dominance of heteromeric GluA1/GluA2 receptors. Thus, α2δ-1 is a pivotal AMPAR-interacting protein that controls the subunit composition and Ca²⁺ permeability of postsynaptic AMPARs.

A linear gating mechanism links kinetically and structurally distinct closed and open states of NMDA receptors. During allosteric inhibition, agonist binding incudes uncoupling of structural changes from gating motions in the first transmembrane region. N-Methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single-molecule Förster resonance energy transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions. These results show kinetically and structurally distinct changes associated with apo, agonist-bound, and inhibited receptors linked by a linear mechanism of gating at this site. Furthermore, the smFRET data suggest that allosteric inhibition by zinc occurs by an uncoupling of the agonist-induced changes at the extracellular domains from the gating motions leading to an apo-like state, while dizocilpine, a pore blocker, stabilizes multiple closely packed transmembrane states.

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) markedly enhance AMPAR function, altering ligand efficacy and receptor gating kinetics and thereby shaping the postsynaptic response. The structural mechanism underlying TARP effects on gating, however, is unknown. Here we find that the prototypical member of the TARP family, stargazin or γ-2, rescues gating deficits in AMPARs carrying mutations that destabilize the closed-cleft states of the ligand-binding domain (LBD), suggesting that stargazin reverses the effects of these mutations and likely stabilizes closed LBD states. Furthermore, stargazin promotes a more closed conformation of the LBD, as indicated by reduced accessibility to the large antagonist NBQX. Consistent with the functional studies, luminescence resonance energy transfer experiments directly demonstrate that the AMPAR LBD is on average more closed in the presence of stargazin, in both the apo and agonist-bound states. The additional cleft closure and/or stabilization of the more closed-cleft states of the LBD is expected to translate to higher agonist efficacy and could contribute to the structural mechanism for stargazin modulation of AMPAR function.