(1) The subunit assembly mechanism and architecture of the ionotropic glutamate receptors.
Ionotropic glutamate receptors are ligand-gated ion channel that are critical for excitatory neurotransmission. They are divided into three subtypes (AMPA, NMDA and kainite receptors) based on their pharmacological characteristics. The heteroterameric AMPA receptors play pivotal roles in synaptic plasticity. Their dysfunction is related to a variety of psychiatric and neurological disorders, including schizophrenia, Alzheimer’s disease, ALS, X-linked mental retardation, limbic encephalitis, CNS lupus, and Rasmussen’s encephalitis.
The exact function and trafficking of these receptors depends critically on their subunit composition and organization. However, because of the limited structural information available on native full-length AMPA receptors, the molecular basis for the function, trafficking, and biogenesis of AMPA receptors remains poorly understood. We study the subunit assembly mechanism and the structures of fully assembled AMPA receptors as well as their assembly intermediates. Recently, we determined the single particle EM structures of the dimeric subunit assembly intermediates of the AMPA-Rs. Our results suggest that specific global domain arrangement of the dimer intermediate is required for the efficient tetramerization. Furthermore, a global domain arrangement in the tetrameric AMPA-R was proposed from this study.
Our ultimate goal is to identify the structural basis for the function and modulation of AMPA receptors. By investigating recombinant AMPA receptors and genetic variants, we aim to extend our previous electron microscopy studies of brain-derived AMPA receptors. Our research further extends into understanding the molecular assembly and function of NMDA receptors. The precise knowledge of the molecular mechanism of ionotropic glutamate receptor function will pave the path toward developing new drugs for treating a variety of neurological and psychiatric disorders.
(2) The function of the NTD of ionotropic glutamate receptors.
The extracellular domain of the ionotropic glutamate receptors consists of two subdomains; the N-terminal domain (NTD) and the ligand-binding domain (LBD). The NTD is the largest domain of the receptor, yet its function is unclear. For the NMDA-Rs the NTD of the GluN2 subunits regulate channel gating by binding to zinc ions. For other subunits the NTD function is largely unknown. Recently, we revealed the X-ray crystal structure and the function of the NTD of GluN1 subunit of NMDA-R. The NTD of GluN1 play critical role during the subunit assembly of the functional heterotetrameric NMDA-Rs.
We propose a model in which initial homodimerization and subsequent rupture of the homodimer are the determinant during the subunit assembly. The new binding cleft identified in this study highlights the potential of this domain as a drug target for future therapeutic agents to cure a variety of neurological and psychiatric diseases.
(3) AMPA and kainite receptor interactomes facilitate identifying novel functional repertoire of iGluRs
In mammalian brain, the majority of excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs), which are ligand-gated ion channels located in the postsynaptic density. The iGluRs are protein complexes formed of tetrameric assembly of core receptor subunits and auxiliary transmembrane subunits. In the case of AMPA-Rs the auxiliary (and candidate auxiliary) subunits include, stargazin/TARPs, SOL-1, cornichon, CKAMP44/Shisa-9, and synDIG1. Each auxiliary subunit modulates channel trafficking and gating is specific ways. The functional variety of AMPA-Rs is therefore amplified by combinatorial effect caused by different types of AMPA-Rs binding to distinct auxiliary subunits. In other words, depending on the type of auxiliary subunits it is associated with, the function of AMPA-R will vary in the brain. The functional variety of iGluR created in this manner may diversify the excitatory modulation in the neural circuits.
The magnitude of molecular variety of iGluR auxiliary (or candidate auxiliary) subunit remains elusive. To gain insight into this question, we have recently conducted a comparative interactome analyses of AMPA and kainite receptors purified from rat brain (Shanks, Savas, Maruo et al. 2012, Cell Reports). With the aid of this large-scale data we were able to identify many candidate auxiliary subunits and/or potential binding partners of AMPA-R and kainite receptors. Among those candidates we have verified GSG1L as novel AMPA-R auxiliary subunit, based on experimental verification by combining methods in biochemistry, electrophysiology and cell biology. In-depth analyses of the biology revolving around GSG1L and further investigation of other potential iGluR interacting membrane proteins identified in our comparative interactome data may reveal novel physiological functions of AMPA-Rs.
(4) The structure and function of molecules that undergo trans-synaptic interactions.
Communication between the pre and postsynaptic neurons are also mediated by the trans-synaptic physical contacts between membrane proteins in the pre and postsynaptic membrane. Examples of proteins in this category are, the cell adhesion molecules (such as cadherins, neurexins-neuroligin, etc..) and the molecules involved in receptor tyrosine kinase signaling (such as Eph receptors and ephrins).
Elucidating the precise molecular mechanism and function of these trans-synaptic molecules is critical for understanding the synaptic function and the topological changes of the neural circuits. Dysfunction of trans-synaptic molecular interactions results in autism spectrum of disorders, such as seen in patients with mutation in the neuroligin and neurexin gene. To contribute to this area of neuroscience research we use structural biological methods to understand the ultrastructures of the trans-synaptic molecular structures in the synapse. Recently, our laboratory determined the single particle EM structure of the extracellular domain of the alpha-neurexin.
(5) Isolation of novel macromolecular complexes from the neuronal membrane.
We believe that there are still novel macromolecules in the membrane that play fundamentally important biological function. Using our strength in membrane biochemistry, we develop new biochemical procedures to isolate new macromolecules from the neuronal membrane. Our interest is not only limited to prototypical transmembrane proteins but also to other molecular entities such as lipid clusters, glycolipid complex, and RNAs. This high-risk high-reward project is partly funded by the NIH EUREKA (Exceptional and Unconventional Research Enabling Knowledge Acceleration) Grant