The Tarantula Venom Peptide Eo1a Binds to the Domain II S3-S4 Extracellular Loop of Voltage-Gated Sodium Channel NaV1.8 to Enhance Activation

Venoms from cone snails and arachnids are a rich source of peptide modulators of voltage-gated sodium (NaV) channels, however relatively few venom-derived peptides with activity at the mammalian NaV1.8 subtype have been isolated. Here, we describe the discovery and functional characterisation of β-theraphotoxin-Eo1a, a peptide from the venom of the Tanzanian black and olive baboon tarantula Encyocratella olivacea that modulates NaV1.8. Eo1a is a 37-residue peptide that increases NaV1.8 peak current (EC50 894 ± 146 nM) and causes a large hyperpolarising shift in both the voltage-dependence of activation (ΔV50–20.5 ± 1.2 mV) and steady-state fast inactivation (ΔV50–15.5 ± 1.8 mV). At a concentration of 10 μM, Eo1a has varying effects on the peak current and channel gating of NaV1.1–NaV1.7, although its activity is most pronounced at NaV1.8. Investigations into the binding site of Eo1a using NaV1.7/NaV1.8 chimeras revealed a critical contribution of the DII S3-S4 extracellular loop of NaV1.8 to toxin activity. Results from this work may form the basis for future studies that lead to the rational design of spider venom-derived peptides with improved potency and selectivity at NaV1.8.

Anti-HNA-3a Alloantibodies Can Cause Differential Endothelial Damage through a Direct Neutrophil Activation Pathway

INTRODUCTION: The human neutrophil antigen (HNA) 3a/b is associated with a single amino acid substitution (R 154Q polymorphism) on the first extracellular loop of choline transporter-like protein 2 (CTL2). Antibodies against HNA-3a have been associated with severe and fatal transfusion-related acute lung injury (TRALI). Epitope mapping suggests the existence of two types of HNA-3a antibodies. Type I antibodies only require first extracellular loop of the CTL2 for expression of the antigen, type II antibodies require at least the first 3 extracellular loops of CTL2 in a native configuration (i.e. expressed on a cell membrane). This study aimed to evaluate the activity of type I and type II anti-HNA-3a antibodies in an in vitro TRALI model. STUDY DESIGN & METHODS: The granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) were used to confirm anti-HNA-3a activity in two donor sera (Q49 and Q50). Flow cytometry was used to confirm antibody binding to freshly isolated human or mouse neutrophils. A rabbit polyclonal antibody against an epitope in the third extracellular loop of CTL2's was coated onto the wells of an ELISA plate and neutrophil HNA-3a antigen was captured and sera tested. For the TRALI model, human lung microvascular endothelial cells (HLMVECs) were grown to confluence before being treated with lipopolysaccharide (LPS). Freshly isolated neutrophils from HNA-3aa homozygote donors were then added with 10% Q49 or Q50. Microscopic counting of Trypan blue staining was used to measure rate HLMVEC death. Significance was determined using a one-way ANOVA (p<0.05), followed by Dunnett's post-hoc test. RESULTS AND DISCUSSION: GAT and GIFT confirmed that both Q49 and Q50 contained an anti-HNA-3a antibody, and that neither contained antibodies against any other HNA or HLA molecules. In flow cytometry, both Q49 and Q50 bound to human neutrophils, but only Q49 bound to mouse neutrophils. In the ELISA, signal was detected only for Q49. These data provided evidence that Q49 was a type I antibody and Q50 was a type II antibody. Within the TRALI model, HLVMEC death was observed with both Q49 and Q50 only in the presence of LPS and neutrophils (Table 1). Previously reported HLMVEC permeability in the absence of neutrophils (Bayat et al. Arterioscler Thromb Vasc Biol. 2013) was not observed as in this study the outcome measured was HLMVEC death. In the present study, the effect for Q49 (type I) was significantly larger than for Q50 (type II; Table 1; p-value <0.0001). CONCLUSIONS: In this model, LPS and neutrophils were required for anti-HNA-3a mediated HLMVEC death thought to reflect events involved in the initiation of TRALI. The observation that type I antibodies elicited a stronger response provides a direction for further research. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Extracellular loop 2 of G protein-coupled olfactory receptors is critical for odorant recognition

G protein-coupled olfactory receptors (ORs) enable us to detect innumerous odorants. They are also ectopically expressed, emerging as attractive drug targets. ORs can be promiscuous or highly specific, which is part of Nature′s strategy for odor discrimination. This work demonstrates that the extracellular loop 2 (ECL2) plays critical roles in OR promiscuity and specificity. Using site-directed mutagenesis and molecular modeling, we constructed 3D OR models in which ECL2 forms a lid of the orthosteric pocket. ECL2 controls the shape and the volume of the odorant-binding pocket, maintains the pocket hydrophobicity and acts as a gatekeeper of odorant binding. The interplay between the specific orthosteric pocket and the variable, less specific ECL2 controls OR specificity and promiscuity. The 3D models enabled virtual screening of new OR agonists and antagonists, exhibiting 78% hit rate in cell assays. This approach can be generalized to structure-based ligand screening for other GPCRs that lack high-resolution 3D structures.

Possible Interactions of Extracellular Loop IVP2-S6 With Voltage-Sensing Domain III in Cardiac Sodium Channel

Motion transmission from voltage sensors to inactivation gates is an important problem in the general physiology of ion channels. In a cryo-EM structure of channel hNav1.5, residues N1736 and R1739 in the extracellular loop IVP2-S6 approach glutamates E1225 and E1295, respectively, in the voltage-sensing domain III (VSD-III). ClinVar-reported variants E1230K, E1295K, and R1739W/Q and other variants in loops IVP2-S6, IIIS1-S2, and IIIS3-S4 are associated with cardiac arrhythmias, highlighting the interface between IVP2-S6 and VSD-III as a hot spot of disease mutations. Atomic mechanisms of the channel dysfunction caused by these mutations are unknown. Here, we generated mutants E1295R, R1739E, E1295R/R1739E, and N1736R, expressed them in HEK-293T cells, and explored biophysical properties. Mutation E1295R reduced steady-state fast inactivation and enhanced steady-state slow inactivation. In contrast, mutation R1739E slightly enhanced fast inactivation and attenuated slow inactivation. Characteristics of the double mutant E1295R/R1739E were rather similar to those of the wild-type channel. Mutation N1736R attenuated slow inactivation. Molecular modeling predicted salt bridging of R1739E with the outermost lysine in the activated voltage-sensing helix IIIS4. In contrast, the loss-of-function substitution E1295R repelled R1739, thus destabilizing the activated VSD-III in agreement with our data that E1295R caused a depolarizing shift of the G-V curve. In silico deactivation of VSD-III with constraint-maintained salt bridge E1295-R1739 resulted in the following changes: 1) contacts between IIIS4 and IVS5 were switched; 2) contacts of the linker-helix IIIS4-S5 with IVS5, IVS6, and fast inactivation tripeptide IFM were modified; 3) contacts of the IFM tripeptide with helices IVS5 and IVS6 were altered; 4) mobile loop IVP2-S6 shifted helix IVP2 that contributes to the slow inactivation gate and helix IVS6 that contributes to the fast inactivation gate. The likelihood of salt bridge E1295-R1739 in deactivated VSD-III is supported by Poisson–Boltzmann calculations and state-dependent energetics of loop IVP2-S6. Taken together, our results suggest that loop IVP2-S6 is involved in motion transmission from VSD-III to the inactivation gates.

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE

SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.

The Role of the Second Extracellular Loop of Norepinephrine Transporter, Neurotrophin-3 and Tropomyosin Receptor Kinase C in T Cells: A Peripheral Biomarker in the Etiology of Schizophrenia

The neurobiology of schizophrenia is multifactorial, comprising the dysregulation of several biochemical pathways and molecules. This research proposes a peripheral biomarker for schizophrenia that involves the second extracellular loop of norepinephrine transporter (NEText), the tropomyosin receptor kinase C (TrkC), and the neurotrophin-3 (NT-3) in T cells. The study of NEText, NT-3, and TrkC was performed in T cells and plasma extracted from peripheral blood of 54 patients with schizophrenia and 54 healthy controls. Levels of NT-3, TrkC, and NET were significantly lower in plasma and T cells of patients compared to healthy controls. Co-immunoprecipitation (co-IPs) showed protein interactions with Co-IP NEText–NT-3 and Co-IP NEText–TrkC. Computational modelling of protein–peptide docking by CABS-dock provided a medium–high accuracy model for NT-3–NEText (4.6935 Å) and TrkC–NEText (2.1365 Å). In summary, immunocomplexes reached statistical relevance in the T cells of the control group contrary to the results obtained with schizophrenia. The reduced expression of NT-3, TrkC, and NET, and the lack of molecular complexes in T cells of patients with schizophrenia may lead to a peripheral dysregulation of intracellular signaling pathways and an abnormal reuptake of norepinephrine (NE) by NET. This peripheral molecular biomarker underlying schizophrenia reinforces the role of neurotrophins, and noradrenergic and immune systems in the pathophysiology of schizophrenia.

Structural insights into hormone recognition by the human glucose-dependent insulinotropic polypeptide receptor

Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone that exerts crucial metabolic functions by binding and activating its cognate receptor, GIPR. As an important therapeutic target, GIPR has been subjected to intensive structural studies without success. Here, we report the cryo-EM structure of the human GIPR in complex with GIP and a Gs heterotrimer at a global resolution of 2.9 Å. GIP adopts a single straight helix with its N terminus dipped into the receptor transmembrane domain (TMD), while the C-terminus is closely associated with the extracellular domain and extracellular loop 1. GIPR employs conserved residues in the lower half of the TMD pocket to recognize the common segments shared by GIP homologous peptides, while uses non-conserved residues in the upper half of the TMD pocket to interact with residues specific for GIP. These results provide a structural framework of hormone recognition and GIPR activation.

Ligand-binding and -scavenging of the chemerin receptor GPR1

Tight regulation of cytokines is essential for the initiation and resolution of inflammation. Chemerin, a mediator of innate immunity, mainly acts on chemokine-like receptor 1 (CMKLR1) to induce the migration of macrophages and dendritic cells. The role of the second chemerin receptor, G protein-coupled receptor 1 (GPR1), is still unclear. Here we demonstrate that GPR1 shows ligand-induced arrestin3 recruitment and internalization. The chemerin C-terminus triggers this activation by folding into a loop structure, binding to aromatic residues in the extracellular loops of GPR1. While this overall binding mode is shared between GPR1 and CMKLR1, differences in their respective extracellular loop 2 allowed for the design of the first GPR1-selective peptide. However, our results suggest that ligand-induced arrestin recruitment is not the only mode of action of GPR1. This receptor also displays constitutive internalization, which allows GPR1 to internalize inactive peptides efficiently by an activation-independent pathway. Our results demonstrate that GPR1 takes a dual role in regulating chemerin activity: as a signaling receptor for arrestin-based signaling on one hand, and as a scavenging receptor with broader ligand specificity on the other. Graphic abstract

Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2

Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are essential for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that mutation of these residues of ECL2 ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.