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NMDA receptor diversity: from molecular dynamics to synaptic physiopathology

Periodic Reporting for period 2 - NMDADYN (NMDA receptor diversity: from molecular dynamics to synaptic physiopathology)

Reporting period: 2018-05-01 to 2019-10-31

NMDA receptors (NMDARs) are a class of neurotransmitter receptors that play critical role in brain development and plasticity. Recent studies have revealed that these glutamate-gated ion channels are more complex than initially thought, undergoing tight subunit-specific regulation by an array of endogenous modulators and existing as multiple subtypes, each with its own anatomical, functional and signaling properties. Such complexity raises key questions regarding the conformational changes that this multi-domain receptor undergoes, the physiological relevance of its subunit plurality and the microenvironment’s impact on receptor and circuit function.

The objective of this multi-scale project is to create and implement the spatially and temporally sensitive tools required to break the barriers to our understanding of NMDAR diversity and modulation. To address these challenges, we are using and developing innovative strategies at the crossroads of protein engineering, biological chemistry and neuroscience to achieve a molecular level control of NMDARs that is subunit-specific, reversible and usable both in vitro and in vivo. Using a bottom-up approach, the project contains four aims covering molecular, cellular and behavioral levels. The first two investigate NMDAR structural mechanisms and exploit this knowledge to develop new optochemical receptor tools. The next two address physiological questions using these tools as well as novel mouse lines.

Aim 1: Characterize NMDAR conformational dynamics and allosteric transitions
Aim 2: Engineer a family of light-controlled NMDARs (‘Opto-allostery’)
Aim 3: Understand the role of specific NMDAR populations in neuronal functions
Aim 4: Explore the receptor’s synaptic microenvironment in normal and disease states

Overall, we expect to provide fundamental insights into the intricate workings of an essential class of brain receptors and further our comprehension of neuronal excitatory transmission in normal and pathological conditions.
Key advances have been made on the following topics:

- NMDAR structural dynamics and allosteric mechanisms
NMDARs form large heterotetrameric molecular complexes. A distinguishing feature of NMDARs is their high conformational mobility. This underlies their remarkable allosteric potential, and may even allow for direct conformational coupling between the extracellular and intracellular domains, independent of pore opening. Though recent atomic structures have provided an excellent structural framework, understanding the plasticity and the functional coupling of the various domains remains a major challenge. Critically, the long-distance allosteric coupling between the membrane distal N-terminal domains (NTDs) and the downstream gating machinery, >100 Ang apart, remained ill defined. Using a combination of experimental approaches (disulfide cross-linking and cellular electrophysiology) and in silico analysis (Normal Mode Analysis), we identified a rotation motion (or rolling) at the interface between the two constitute dimers as a key structural mechanism in NMDAR activation and allosteric modulation. Our single-channel kinetics analysis reveals that this reorientation of the two dimers precipitates pre-open channels to switch into the action open state. Moreover, we established that inter-dimer rolling provides a conformational route by which structural changes within the NTD layer are transduced into rearrangement of the downstream gating machinery. Altogether, our results provide the first integrated views of long-distance domains coupling and dynamics in a full-length NMDAR. It also highlights the potential of glutamate receptor inter-dimer interfaces as novel sites for pharmacological manipulations.

- Molecular optogenetics and NMDAR functional diversity
Reprogramming receptor to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Light confers high spatio-temporal resolution and combines to genetics and pharmacology permit a molecular and cellular control level with unrivaled precision. During the last two years and a half, we have designed a set of light controllable NMDARs using either photosensitive amino acids or ligands.
In a first work, we provide the first demonstration of real-time detection of molecular rearrangements due to reversible light-switching of single amino acid side-chains inserted into a neuronal receptor. To achieve this, we genetically incorporated azobenzene-based photoswitchable amino acids (PSAAs) into NMDARs. We show using optically-controlled patch-clamp recordings that PSAA incorporation occurs with high fidelity and endows robust photoregulation combining full reversibility, bi-directionality, high temporal precision, and molecular (subunit) specificity. By introducing PSAA at various locations in the large multidomain receptor, we were able to photocontrol channel open probability, agonist sensitivity, and ion permeation. These results establish the utility of single photo-active amino acids to directly control transmembrane domain motions and ion channel properties.
In a second and still ongoing work, we developed new photoswitchable tethered ligands (PTLs) capable of reversibly binding subunit-specific sites on NMDARs to regulate their activity. We thus chemically designed photoswitchable allosteric potentiators for GluN2B-reeptors exploiting the GluN2B-selective polyamine modulatory site. We identified several sites on the GluN2B N-terminal domain for PTL attachment and producing reversible and precisely controlled light-mediated regulation of GluN2B-NMDAR activity. The method with its pharmacological specificity and genetic encodability has the major advantage of limiting off-target effects because the photoswitchable ligand can be directly attached to the target receptor. We have now obtained a full data set on heterologously expressed recombinant receptors demonstrating the feasibility and viability of the approach. W
1. We have set-up a dual strategy based on the combination of experimental and computational approaches to decipher the molecular mechanism and structural dynamics of NMDARs. With the ongoing implementation of the voltage-clamp fluorometry approach, we expect to provide a comprehensive view on how these multi-domain multi-subunit receptor complexes operate at the molecular and structural level.

2. Using innovative optopharmacological approaches, we have designed a set of precise light-controllable NMDAR subunits. We are now translating these receptor tools into native settings to decode the role of specific NMDAR populations in neuronal networks.

3. We have discovered that neurons express a new family NMDA receptors activated by glycine only and composed of the GluN3A subunit (glycine excitatory GluN1/GluN3A receptors). This discovery has broad ranging implications for the study and exploration of GluN3A receptors in brain development and function.

4. We have generated original mouse lines (ZnT3-Cre, condition GluN1 glycine/D-serine knock-in) that are expected to provide essential novel information on the composition, plasticity and functional importance of the synaptic microenvironment.
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