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 the macromolecular machinery is frequently associated with neurological disorders such as schizophrenia, epilepsy, stroke, depression, Parkinson’s disease, and Alzheimer’s disease, which 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. We are also recently interested in biology at the neuron-cancer interface. Our studies involve 1) biochemistry and structural biology of membrane-embedded or membrane-bound receptors and signaling molecules that interact with them; 2) functional characterization of ion channels by electrophysiology; 3) protein engineering of reagents such as antibodies and artificial protein probes; 4) experimental and computation-based compound development; and 5) imaging of cellular complexes by light and electron microscopes.

Techniques we use

The research in the Furukawa lab employs multidisciplinary approaches to answer biological questions. They include structural biology, electrophysiology, biophysics, cell biology, protein engineering, and small compound development. Two major structural biological methods employed are x-ray crystallography and single-particle electron cryo-microscopy (cryo-EM). We also implement MD simulation to elaborate our results from the experimental structural biology and pharmacology. Various forms of electrophysiology including patch-clamp and two-electrode-voltage-clamp 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 and proteases may be made and engineered by synthetic biological approaches such as yeast/phage-display technology coupled with directed protein evolution. To facilitate all aspects of the studies, we often tweak and develop new methods. For example, we recently identified the optimal combination of UTRs and promoters for the expression and assembly of multimeric membrane protein complexes (EarlyBac). 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:

Structure and Function of intact NMDA receptor ion channels

NMDA receptors are heterotetrameric cationic ion channels that are composed of two GluN1 subunits and two GluN2 (A-D) and/or GluN3 (A-B) subunits. GluN1 and GluN3 subunits bind glycine whereas GluN2 subunits bind the neurotransmitter, glutamate. Concurrent binding of glycine and glutamate is necessary for the opening of the cationic ion channel, which contributes to the generation of excitatory postsynaptic potential (epsp). Furthermore, voltage-dependent calcium influx by NMDA receptors drives cellular signaling for neuroplasticity, which is critical for learning and memory formation. Dysfunctional NMDA receptors are implicated in Alzheimer’s disease, Parkinson’s disease, depression, schizophrenia, stroke, and autism. We solved the crystal structure of the intact heterotetrameric GluN1-GluN2B NMDA receptors from rat in complex with glycine, glutamate, and an allosteric inhibitor, ifenprodil. This structure revealed how the extracellular domains, amino-terminal domains (ATDs), and ligand-binding domains (LBDs), 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 single-particle electron cryo-microscopy (cryo-EM), we attempt to understand the 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 in our building allows us to explore the above at high quality. In combination with electrophysiology and MD simulation, we have been addressing functional questions including allosteric modulations, pH-sensitivity mediated by alternative splicing, activation, and competitive inhibition.

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. NMDA receptors are diverse in that eight splicing variants of GluN1 can form heterotetramers with four different types of GluN2s (A-D) and two different types of GluN3s (A-B) to form ion channels with completely different functions (e.g. open probability, the kinetics of opening/closing, compound binding, and potency). Subtype-specific compounds/reagents against NMDA receptors would be effective against neurological diseases and disorders mentioned above, thus, understanding the key elements for subtype-specificity at high-resolution is important. While we implement cryo-EM to obtain the structure of the intact heterotetrameric NMDA receptors to obtain medium-range resolution structures (3 – 6 angstrom), we employ x-ray crystallography on the two extracellular domains, an amino-terminal domain (ATD) and a ligand-binding domain (LBD), which bind allosteric modulators and neurotransmitter agonists, respectively, in complex with compounds of interest to obtain high-resolution structures (2.5 angstroms and higher) in order to visualize compounds, critical water molecules, and conformational alterations within the domains. 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.

Large-pore channels

Biological membranes of many tissues and organs contain large pore channels designed to permeate a wide variety of ions and metabolites such as ATP. Examples of those channels include the connexin, innexin, and pannexin channel families, members of which form gap junctions and/or bona fide cell surface channels. These pores rely on a manifold of regulatory means such as voltage-gating for cellular homeostasis and viability and mediate a wide variety of biological processes. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), which permeate small ions and ATP in a voltage-dependent manner to mediate various physiological processes including modulation of neuronal excitability, taste signaling, as well as pathologies of depression and Alzheimer’s disease. However, despite such critical biological roles, the structure and pattern of oligomeric assembly remain unclear. We implement cryo-EM and electrophysiology to understand how CALHM proteins assemble and mediate the permeation of various ions and metabolites in a voltage-dependent manner. We also attempt to understand another unrelated large pore channel, Pannexin, involved in ATP efflux in collaboration with the Kawate lab at Cornell University.

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 by basic immunization technology and/or 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.