Emerging insights into the structure and function of ionotropic glutamate delta receptors.

Glutamate delta-1 (GluD1) and delta-2 (GluD2) receptors belong to the orphan GluD subfamily of ionotropic glutamate receptors (iGluRs). GluDs were classified as ionotropic glutamate receptors based on their sequence similarity. Two decades after these GluDs were first cloned they are still considered "orphan" due to a lack of knowledge of the endogenous ligands that can activate them. Nevertheless, they are crucial for synapse formation, maturation and maintenance of CNS functions, and are implicated in multiple neuronal disorders, including schizophrenia, autism spectrum disorder and depressive disorders. Over the last decade significant discoveries have been made, include role of GluD receptors in mediating trans-synaptic interactions and their unique non-swapped architecture, which is distinct from other ionotropic glutamate receptors. Also, the prospect of GluD ionotropic activity being regulated by direct interaction with metabotropic glutamate receptors is exciting. These discoveries will likely drive the field in the future, providing direction to GluD research. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Role of Neto1 extracellular domain in modulation of kainate receptors.

Kainate receptors play fundamental roles in regulating synaptic transmission and plasticity in central nervous system and are regulated by their cognate auxiliary subunits Neuropilin and tolloid-like proteins 1 and 2 (Neto). While electrophysiology-based insights into functions of Neto proteins are known, biophysical and biochemical studies into Neto proteins have been largely missing till-date. Our biochemical, biophysical, and functional characterization of the purified extracellular domain (ECD) of Neto1 shows that Neto1-ECD exists as monomers in solution and has a micromolar affinity for GluK2 receptors in apo state or closed state. Remarkably, the affinity was ~2.8 fold lower for receptors trapped in the desensitized state, highlighting the conformation-dependent interaction of Neto proteins with kainate receptors. SAXS analysis of Neto1-ECD reveals that their dimensions are long enough to span the entire extracellular domain of kainate receptors. The shape and conformation of Neto1-ECD seems to be altered by calcium ions pointing towards its possible role in modulating Neto1 functions. Functional assays using GluK2 receptors and GluK2/GluA2 chimeric receptors reveal a differential role of Neto1 domains in modulating receptor functions. Although the desensitization rate was not affected by the Neto1-ECD, the recovery rates from the desensitized state are altered.

Structural biology of ionotropic glutamate delta receptors and their crosstalk with metabotropic glutamate receptors.

Enigmatic orphan glutamate delta receptors (GluD) are one of the four classes of the ionotropic glutamate receptors (iGluRs) that play key roles in synaptic transmission and plasticity. While members of other iGluR families viz AMPA, NMDA, and kainate receptors are gated by glutamate, the GluD receptors neither bind glutamate nor evoke ligand-induced currents upon binding of glycine and D-serine. Thus, the GluD receptors were considered to function as structural proteins that facilitate the formation, maturation, and maintenance of synapses in the hippocampus and cerebellum. Recent work has revealed that GluD receptors have extensive crosstalk with metabotropic glutamate receptors (mGlus) and are also gated by their activation. The latest development of a novel optopharamcological tool and the cryoEM structures of GluD receptors would help define the molecular and chemical basis of the GluD receptor's role in synaptic physiology. This article is part of the special Issue on "Glutamate Receptors - Orphan iGluRs".

The architecture of GluD2 ionotropic delta glutamate receptor elucidated by cryo-EM.

GluD2 receptor belongs to the orphan delta family of glutamate receptor ion channels. These receptors play key roles in synaptogenesis and synaptic plasticity and are associated with multiple neuronal disorders like schizophrenia, autism spectrum disorder, cerebellar ataxia, intellectual disability, paraplegia, retinal dystrophy, etc. Despite the importance of these receptors in CNS, insights into full-length GluD2 receptor structure is missing till-date. Here we report cryo-electron microscopy structure of the rat GluD2 receptor in the presence of calcium ions and the ligand 7-chlorokynurenic acid, elucidating its 3D architecture. The structure reveals a non-swapped architecture at the extracellular amino-terminal (ATD), and ligand-binding domain (LBD) interface similar to that observed in GluD1; however, the organization and arrangement of the ATD and LBD domains in GluD2 are unique. While our results demonstrate that non-swapped architecture is conserved in the delta receptor family, they also highlight the differences that exist between the two member receptors; GluD1 and GluD2.

Structural dynamics of the GluK3-kainate receptor neurotransmitter binding domains revealed by cryo-EM.

Kainate receptors belong to the ionotropic glutamate receptor family and play critical roles in the regulation of synaptic networks. The kainate receptor subunit GluK3 has unique functional properties and contributes to presynaptic facilitation at the hippocampal mossy fiber synapses along with roles at the post-synapses. To gain structural insights into the unique functional properties and dynamics of GluK3 receptor, we imaged them via electron microscopy in the apo-state and in complex with either agonist kainate or antagonist UBP301. Our analysis of all the GluK3 full-length structures not only provides insights into the receptor transitions between desensitized and closed states but also reveals a "non-classical" conformation of neurotransmitter binding domain in the closed-state distinct from that observed in AMPA and other kainate receptor structures. We show by molecular dynamics simulations that Asp759 influences the stability of the LBD dimers and hence could be responsible for the observed conformational variability and dynamics of the GluK3 via electron microscopy. Lower dimer stability could explain faster desensitization and low agonist sensitivity of GluK3. In overview, our work helps to associate biochemistry and physiology of GluK3 receptors with their structural biology and offers structural insights into the unique functional properties of these atypical receptors.

Cryo-EM structures of the ionotropic glutamate receptor GluD1 reveal a non-swapped architecture.

Ionotropic orphan delta (GluD) receptors are not gated by glutamate or any other endogenous ligand but are grouped with ionotropic glutamate receptors (iGluRs) based on sequence similarity. GluD1 receptors play critical roles in synaptogenesis and synapse maintenance and have been implicated in neuronal disorders, including schizophrenia, cognitive deficits, and cerebral ataxia. Here we report cryo-EM structures of the rat GluD1 receptor complexed with calcium and the ligand 7-chlorokynurenic acid (7-CKA), elucidating molecular architecture and principles of receptor assembly. The structures reveal a non-swapped architecture at the interface of the extracellular amino-terminal domain (ATD) and the ligand-binding domain (LBD). This finding is in contrast with structures of other families of iGluRs, where the dimer partners between the ATD and LBD layers are swapped. Our results demonstrate that principles of architecture and symmetry are not conserved between delta receptors and other iGluRs and provide a molecular blueprint for understanding the functions of the 'orphan' class of iGluRs.

Structural and Functional Insights into GluK3-kainate Receptor Desensitization and Recovery.

GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play a vital role in the regulation of synaptic transmission. For a better understanding of structural changes that underlie receptor functions, GluK3 receptors were trapped in desensitized and resting/closed states and structures analyzed using single particle cryo-electron microscopy. While the desensitized GluK3 has domain organization as seen earlier for another kainate receptor-GluK2, antagonist bound GluK3 trapped a resting state with only two LBD domains in dimeric arrangement necessary for receptor activation. Using structures as a guide, we show that the N-linked glycans at the interface of GluK3 ATD and LBD likely mediate inter-domain interactions and attune receptor-gating properties. The mutational analysis also identified putative N-glycan interacting residues. Our results provide a molecular framework for understanding gating properties unique to GluK3 and exploring the role of N-linked glycosylation in their modulation.

Improved neural differentiation of normal and abnormal induced pluripotent stem cell lines in the presence of valproic acid.

During the generation of induced pluripotent stem cell (iPSC) lines from cord blood CD34+ cells, a line having complete trisomy of Chromosome 1 and deletion of q23 to qTer of Chromosome 11 was accidentally developed in our lab. The abnormality was consistently detected even at higher passages. These chromosomal anomalies are known to manifest neurological developmental defects. In order to examine if such defects occur during in vitro differentiation of the cell line, we set up a protocol for neural differentiation. Valproic acid (VPA) was earlier reported by us to enhance neural differentiation of placental mesenchymal stem cells. Here, we induced normal and abnormal iPSC lines to neural lineage with/without VPA. Neural differentiation was observed in all four sets, but for both the iPSCs lines, VPA sets performed better. The characteristics tested were morphology, neural filament length, detection of neural markers, and electrophysiology. In summary, the karyotypically abnormal line exhibited efficient neural differentiation. This iPSC line may serve as a useful tool to study abnormalities associated with trisomy 1 and deletion of q23 to qTer of Chromosome 11.

A comparison of aquaporin function in mediating stomatal aperture gating among drought-tolerant and sensitive varieties of rice (Oryza sativa L.).

Climate change drastically affects the cultivation of rice, and its production is affected significantly by water stress. Adaptation of a plant to water deficit conditions is orchestrated by efficient water uptake and a stringently regulated water loss. Transpiration remains the major means of water loss from plants and is mediated by microscopic pores called stomata. Stomatal aperture gating is facilitated by ion channels and aquaporins (AQPs) which regulate the turgidity of the guard cells. In a similar manner, efficient water uptake by the roots is regulated by the presence of AQPs in the plasma membrane of root cells. In this study, we compare the efficiency of transmembrane water permeability in guard cells and root protoplasts from drought-tolerant and sensitive varieties of Oryza sativa L. In this report, we studied the transmembrane osmotic water permeability (Pos) of guard cell and root protoplasts of drought-sensitive and tolerant cultivars. The guard cells isolated from the drought-sensitive lowland rice variety ADT-39 show significant low osmotic permeability than the drought-tolerant rice varieties of Anna (lowland) and Dodda Byra Nellu (DBN) (upland local land rice). There is no significant difference in relative gene expression patterns of PIPs (Plasma membrane Intrinsic Proteins "PIP1" and "PIP2" subfamilies) in guard cells isolated from ADT-39 and Anna. While the expression levels of AQP genes remain the same between ADT-39 and Anna, there is a drastic difference in their osmotic permeability in the guard cells in spite of a higher number of stomata in Anna and DBN, hinting at a more efficient gating mechanism of AQP in the stomata of the drought-tolerant varieties studied.