Structural biology, biochemistry, and physiology of neuronal signaling

High-order brain functions including learning and memory formation are the results of complex cellular signal transduction events mediated by the assembly of macromolecules in neurons that respond to given environments. Dysfunction of these macromolecular machineries is frequently associated with neurological disorders such as schizophrenia, epilepsy, strike, depression, Parkinson’s disease and Alzheimer’s disease that are challenging and debilitating clinical problems today. Our broad biological interests revolve around the cellular paradigms in neuroscience such as neuroplasticity and neurodegeneration, which are mediated by changes in membrane potentials and numbers of protein-protein interactions. Currently, most of our studies involve neuropharmacology of membrane-embedded or membrane-bound receptors and signaling molecules that interact with them.

Techniques we use

The research in the Furukawa lab takes a multi-disciplinary approach. It includes structural biology, electrophysiology, biophysics, cell biology, protein engineering, and method development. Two major structural biological methods employed are x-ray crystallography and electron cryo-microscopy (cryo-EM). Various forms of electrophysiology are implemented to detect and analyze electrical signals elicited by ion channels and transporters. We measure the affinity of compound-protein interactions or protein-protein interactions using biophysical techniques such as ITC, SPR, and thermophoresis. Novel protein-protein interactions in cells may be identified using approaches involving proteomics in collaboration with our proteomics facility at CSHL. Proteins such as antibodies may be made and engineered in collaboration with our antibody facility specialized in mouse-immunization, llama-immunization as well as yeast/phage-display technology. To facilitate all aspects of the studies, we often develop new methods. For example, we recently identified the optimal combination of UTRs and promoters for expression and assembly of multimeric membrane protein complexes. Hypotheses can be derived from data in any one of the disciplines above and tested by the others. For example, a hypothesis derived from structural biological information can be tested by electrophysiology and biophysics and vice versa. Below are brief descriptions of some of the research projects that are taking place in our group:

High-resolution structural biology of NMDA receptor extracellular domains

Since the discovery of NMDA receptors in the early 1970s, great efforts have been put forth to characterize the NMDA receptor pharmacology. Despite enthusiasm in the field, structural studies of NMDA receptors was slow to develop because they are large multimeric membrane protein complexes with many sites of glycosylation, which are technically difficult to express, purify, and crystallize for x-ray crystallography. For that reason, we worked on the two extracellular domains, an amino terminal domain (ATD) and a ligand-binding domain (LBD), which bind allosteric modulators and neurotransmitter agonists, respectively (Figure). We are continuing to pursue high-resolution crystallography in those domains to identify structural determinants for subtype-specificity, understand the chemical nature of compound binding sites, and help the field of neuropharmacology to improve drug design. Despite the popularity of cryo-EM, precise detection of compound-water-protein interactions at high resolution, which is inevitable for compound development can only be achieved by crystallography at this point. The need for developing subtype-specific inhibitors became obvious since the recent discovery that inhibition of the NMDA receptor signal is effective in treatment of depression. Furthermore, over-flux of calcium into neurons causes excitotoxicity and subsequently neurodegeneration such as Alzheimer’s disease. Also there are many de novo mutations that cause multiple forms of epilepsy. Finally, the NMDA receptor signaling has recently been shown to control invasiveness of tumors. By providing structural tools to visualize drug binding sites at high-resolution, we are hoping to facilitate the field of pharmacology to create effective therapeutic compounds.

Structural biology of intact NMDA receptor ion channels

After extensive efforts, we solved the crystal structure of the intact heterotetrameric GluN1-GluN2B NMDA receptors from rat in 2014. This structure revealed how ATD and LBD from both GluN1 and GluN2 subunits are associated with each other in the ‘dimer of GluN1-GluN2 heterodimers’ manner. By a combination of x-ray crystallography and electron cryo-microscopy (cryo-EM), we attempt to understand regulatory mechanisms of NMDA receptors. Specifically, we are working toward understanding how multiple domains (ATD, LBD, and TMD) move with respect to one another in various functional states. The state-of-the-art cryo-EM facility established in late 2017 now allows us to explore the above at high quality.

Biochemistry and structural biology of signaling molecules involved in neuroplasticity

The calcium influx via NMDA receptor and voltage-gated calcium channels mediate subsequent signaling for plasticity and neurodegeneration. Research in the past showed that kinases such as CaMKII and PKA are heavily involved in the process. We are interested in isolating and characterizing protein complexes involved in cellular signaling of neuroplasticity by biochemical, proteomics, and cell biological methods.

Antibody screening and engineering

Antibodies are extremely useful biological reagents that can recognize and functionally regulate target proteins. We are screening and developing antibodies for cell surface receptors expressed in neurons. This is done in our state-of-the-art antibody facility at CSHL, which utilizes a basic immunization technology and the phage- and yeast-display methods. Identified antibodies are further tested for functional effects and are re-engineered using structural information. We are hoping to develop antibodies that can be used as therapeutic reagents, biomarkers, and tools for basic researches that involve manipulation of neuronal receptors and improvement of structural biological methods.