Designing a family of photoswitchable NMDA receptor subunits to interrogate receptor functional diversity.
Final Report Abstract
Nerve cells communicate with each other by releasing chemicals, also known as neurotransmitters, from one cell to the next. Once released, these neurotransmitters bind to specific docking stations, called receptors, which are located on the surface of the neighboring cell. Due to changes in neurotransmitter release or the receptor number, the connections between neurons can either strengthen or weaken over time. This process, called synaptic plasticity, forms the basis of learning and memory. One of the key players in synaptic plasticity are NMDA receptors (NMDARs), and if these receptors are faulty, it can cause disorders such as schizophrenia or epilepsy. NMDAs are a large family of receptors that have a large variety receptor subtypes, each with specific properties. Every subtype is composed of four varying subunits. It is still unclear how these different receptor subtypes contribute to the different forms of synaptic plasticity. There is a great need for novel approaches allowing to selectively manipulate specific receptor subtypes in defined cell populations with high temporal resolution. The proposed project aimed to design and implement an innovative strategy using azobenzene-based photoswitchable UAAs (PSAAs), which combines the advantages of unnatural amino acid (UAA) incorporation and azobenzene photochemistry, to investigate roles of specific receptor populations. Engineering light-sensitivity into proteins by introduction of azobenzene moieties has wide ranging applications in molecular studies and neuroscience. Upon exposures to light of different wavelengths, azobenzene groups undergo a reversible photoswitching between two different configurations – a compact cis- and a stretched trans-configuration. The use of azobenzenes offers high quantum yield, minimal photo-bleaching, excellent photo-stability, and has the major benefit of functional reversibility. Commonly used tethered photoswitchable ligands, however, require solvent-accessible protein labeling, face structural constrains, and are bulky. Here, we have generated a family of NMDARs by directly incorporating single PSAAs providing genetic encodability, reversibility, and site tolerance. We identified several positions within the multi-domain receptor that allowed to control receptor activity with light. Remarkably, these effects were driven by a minimal photoswitching event involving toggling between the elongated trans- and the bent cis-isoform of single azobenzene side-chains. PSAA introduction within different subunits and receptor domains enabled rapid, stable, and reproducible photo-control. PSAA photo-isomerization within the receptor ABDs allowed control of co-agonist sensitivity and activation efficacy. Strikingly, targeting transmembrane pore sites, whose conformational changes during receptor activity remain poorly understood, enabled optical modulation of gating and permeation properties. Moreover, both subunit-specific and bi-directional (photo-inactivation and photo-potentiation) receptor control were achieved. Our study demonstrates the first detection of molecular rearrangements in real-time due to the reversible light-switching of single amino acid side-chains, adding a dynamic dimension to protein site-directed mutagenesis. This novel approach to interrogate neuronal protein function has general applicability in the fast expanding field of optopharmacology. Further research will use this toolset of PSAA-controllable receptors to study how the different NMDA receptor subtypes affect synaptic plasticity in the normal and diseased brain.
Publications
- (2017) Optocontrol of Glutamate Receptor Activity By Single Side-Chain PhotoisomerizationN. eLife
Klippenstein V, Hoppmann C, Ye S, Wang L, Paoletti P
(See online at https://doi.org/10.7554/eLife.25808.001) - (2018) Probing Ion Channel Structure and Function Using Light-Sensitive Amino AcidsS. Trends in Biochemical Sciences
Klippenstein V, Mony L, Paoletti P
(See online at https://doi.org/10.1016/j.tibs.2018.02.012)
