Single-Molecule FRET Analyses of NMDA Receptors.

Single-molecule fluorescence resonance energy transfer (smFRET) enables the real-time observation of conformational changes in a single protein molecule of interest. These observations are achieved by attaching fluorophores to proteins of interest in a site-specific manner and investigating the FRET between the fluorophores. Here we describe the method wherein the FRET is studied by adhering the protein molecules to a slide using affinity-based interactions and measuring the fluorophores' fluorescence intensity from a single molecule over time. The resulting information can be used to derive distance values for a point-to-point measurement within a protein or to calculate kinetic transition rates between various conformational states of a protein. Comparing these parameters between different conditions such as the presence of protein binding partners, application of ligands, or changes in the primary sequence of the protein can provide insights into protein structural changes as well as kinetics of these changes (if in the millisecond to second timescale) that underlie functional effects. Here we describe the procedure for conducting analyses of NMDA receptor conformational changes using the above methodology and provide a discussion of various considerations that affect the design, execution, and interpretation of similar smFRET studies.

Bi-directional allosteric pathway in NMDA receptor activation and modulation.

N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors involved in learning and memory. NMDA receptors primarily comprise two GluN1 and two GluN2 subunits. The GluN2 subunit dictates biophysical receptor properties, including the extent of receptor activation and desensitization. GluN2A- and GluN2D-containing receptors represent two functional extremes. To uncover the conformational basis of their functional divergence, we utilized single-molecule fluorescence resonance energy transfer to probe the extracellular domains of these receptor subtypes under resting and ligand-bound conditions. We find that the conformational profile of the GluN2 amino-terminal domain correlates with the disparate functions of GluN2A- and GluN2D-containing receptors. Changes at the pre-transmembrane segments inversely correlate with those observed at the amino-terminal domain, confirming direct allosteric communication between these domains. Additionally, binding of a positive allosteric modulator at the transmembrane domain shifts the conformational profile of the amino-terminal domain towards the active state, revealing a bidirectional allosteric pathway between extracellular and transmembrane domains.

Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel.

Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.

Structural Dynamics of Glutamate Signaling Systems by smFRET.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for investigating the structural dynamics of biological macromolecules. smFRET reveals the conformational landscape and dynamic changes of proteins by building on the static structures found using cryo-electron microscopy, x-ray crystallography, and other methods. Combining smFRET with static structures allows for a direct correlation between dynamic conformation and function. Here, we discuss the different experimental setups, fluorescence detection schemes, and data analysis strategies that enable the study of structural dynamics of glutamate signaling across various timescales. We illustrate the versatility of smFRET by highlighting studies of a wide range of questions, including the mechanism of activation and transport, the role of intrinsically disordered segments, and allostery and cooperativity between subunits in biological systems responsible for glutamate signaling.

Allosteric Changes in the NMDA Receptor Associated with Calcium-Dependent Inactivation.

N-methyl-D-aspartate (NMDA) receptors mediate synaptic excitatory signaling in the mammalian central nervous system by forming calcium-permeable transmembrane channels upon binding glutamate and coagonist glycine. Ca2+ influx through NMDA receptors leads to channel inactivation through a process mediated by resident calmodulin bound to the intracellular C-terminal segment of the GluN1 subunit of the receptor. Using single-molecule FRET investigations, we show that in the presence of calcium-calmodulin, the distance across the two GluN1 subunits at the entrance of the first transmembrane segment is shorter and the bilobed cleft of the glycine-binding domain in GluN1 is more closed when bound to glycine and glutamate relative to what is observed in the presence of barium-calmodulin. Consistent with these observations, the glycine deactivation rate is slower in the presence of calcium-calmodulin. Taken together, these results show that the binding of calcium-calmodulin to the C-terminus has long-range allosteric effects on the extracellular segments of the receptor that may contribute to the calcium-dependent inactivation.

Conformational spread and dynamics in allostery of NMDA receptors.

Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer-dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.

Mechanism of modulation of AMPA receptors by TARP-γ8.

Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by glutamate-activated α-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA) receptors. In neurons, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs). Assembly with TARP γ8 alters the biophysical properties of the receptor, producing resensitization currents in the continued presence of glutamate. Using single-channel recordings, we show that under resensitizing conditions, GluA2 AMPA receptors primarily transition to higher conductance levels, similar to activation of the receptors in the presence of cyclothiazide, which stabilizes the open state. To study the conformation associated with these states, we have used single-molecule FRET and show that this high-conductance state exhibits tighter coupling between subunits in the extracellular parts of the receptor. Furthermore, the dwell times for the transition from the tightly coupled state to the decoupled states correlate to longer open durations of the channels, thus correlating conformation and function at the single-molecule level.

Single-Molecule FRET Methods to Study Glutamate Receptors.

Single-molecule fluorescence energy transfer methods allow us to determine the complete structural landscape between the donor and acceptor fluorophores introduced on the protein of interest. This method is particularly attractive to study ion channel proteins as single-molecule current recordings have been used to study the function of these proteins for several decades. Here we describe the smFRET method used to study glutamate receptors.

Mapping the Conformational Landscape of Glutamate Receptors Using Single Molecule FRET.

The ionotropic glutamate receptors mediate excitatory neurotransmission in the mammalian central nervous system. These receptors provide a range of temporally diverse signals which stem from subunit composition and also from the inherent ability of each member to occupy multiple functional states, the distribution of which can be altered by small molecule modulators and binding partners. Hence it becomes essential to characterize the conformational landscape of the receptors under this variety of different conditions. This has recently become possible due to single molecule fluorescence resonance energy transfer measurements, along with the rich foundation of existing structures allowing for direct correlations between conformational and functional diversity.