Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.Nicotinic acetylcholine receptors (nAChRs) belong to the class of pentameric ligand-gated ion channels or Cys-loop receptors, which play a crucial role in fast synaptic signaling in the central and peripheral nervous system (1, 2). nAChRs are specifically activated by the endogenous neurotransmitter acetylcholine, which binds to a ligand-binding site in the extracellular domain of the receptor and triggers opening of a channel gate located downstream in the transmembrane domain. This results in a flux of cations through the channel pore, which depolarizes the cell membrane and initiates the action potential. Together with serotonin 5-HT₃ receptors, nAChRs belong to the category of excitatory ligand-gated ion channels. In contrast, glycine and γ-aminobutyric acid (GABA_A/C) receptors form anion-selective pores and belong to the category of inhibitory ligand-gated ion channels. Cys-loop receptors are mutated in a diverse range of inherited neurological disorders, including epilepsy, myasthenia gravis, and hyperekplexia. Additionally, Cys-loop receptors form the target for a wide variety of therapeutically used drugs, including benzodiazepines, barbiturates, general anesthetics, and anthelmintics (3–5). Important to note is that most of these drugs exert their effect by binding to an allosteric site that is remote from the agonist binding site of the receptor.Most of the high-resolution structural information on nicotinic receptors derives from molluscan acetylcholine binding proteins (AChBPs), which are homologous to the large extracellular ligand-binding domain of nAChRs but lack a pore-forming transmembrane domain (3, 6–9). The architecture of the complete Torpedo nAChR ion channel is shown in Fig. 1A, displaying the extracellular, transmembrane, and intracellular domains. AChBPs assemble as homopentamers and have an architectural fold that closely resembles the ligand-binding domain of Cys-loop receptors, including the invertebrate glutamate-gated chloride channel GluCl (10, 11), as well as the bacterial homologs GLIC (8, 12, 13) and ELIC (8, 14). The pharmacological properties of AChBPs are mostly similar to the neuronal α7 subtype of nicotinic receptors (8, 10, 15, 16), which also function as homopentamers. To date, more than 70 cocrystal structures of AChBPs in complex with different agonists, partial agonists, and antagonists have been determined. These studies have revealed a wealth of information on the structural determinants of ligand recognition and the conformational changes occurring during ligand binding. The orthosteric ligand-binding site is located at the interface between a “principal” and “complementary” subunit and is lined by several highly conserved aromatic residues located on three loops in each subunit, termed loops A, B, and C on the principal face and loops D, E, and F on the complementary face. Ligand binding triggers conformational changes in the ligand-binding site, which couple to channel opening. Despite these insights, structural data on ligand recognition are currently limited to ligands binding in the orthosteric or agonist binding site of nicotinic receptors, whereas information on allosteric binding sites is lacking. Detailed structural knowledge of possible allosteric binding sites in nicotinic receptors is fundamentally important and has great therapeutic potential for the development of allosteric modulators. This is especially clear in the context of benzodiazepines, which bind at allosteric binding sites of the extracellular domain of the GABA_A receptors and are widely prescribed as hypnotics, sedatives, anxiolytics, antiepileptics, and muscle relaxants (17, 18). Positive allosteric modulators (PAMs) of the α7 nAChR are also recognized as having therapeutic potential. Two different types are distinguished based on the functional effect: type I PAMs, for example NS-1738 (17, 19), mainly potentiate the peak current response evoked by the agonist ACh, whereas type II PAMs, for example PNU-120596 (20, 21), both potentiate the peak current response and alter the time course (4, 20). Based on the results of chimeric protein domain fusion experiments distinct allosteric binding sites are expected: Type I PAMs bind at a site in the extracellular domain, whereas type II PAMs bind at a site in the transmembrane domain (20, 22). However, structural data on the molecular determinants of PAM recognition are currently lacking.Open in a separate windowFig. 1.Structure of α7-AChBP and location of the agonist binding site. (A) Side view of the Torpedo nAChR (PDB ID code 2BG9) in cartoon representation. Different colors are used for extracellular domain (ECD, blue), transmembrane domain (TMD, red) and intracellular domain (ICD, green). (B) Top-down view of the α7-AChBP crystal structure in complex with lobeline. Each subunit of the pentamer is shown in a different color. (C) Detailed side view of the agonist binding site in α7-AChBP, which is localized at the subunit interface of the (+) and (−) subunit. Lobeline is shown in yellow sphere representation. The α7-AChBP chimera is shown in cartoon representation and blue regions correspond to residues originating from the α7 nAChR (UniProt accession code {"type":"entrez-protein","attrs":{"text":"P36544","term_id":"2506127"}}P36544), whereas orange regions correspond to residues from Lymnaea AChBP (UniProt accession code {"type":"entrez-protein","attrs":{"text":"P58154","term_id":"14285341"}}P58154). (D and E) Detailed view of the agonist binding site, highlighting the different binding pose of lobeline in the α7-AChBP structure (D) and the Aplysia AChBP structure (E). The conserved aromatic residues of the agonist binding site are shown in ball and stick representation. The blue circles indicate the corresponding ‟loops” of the binding site, termed loops A, B, and C on the (+) subunit and loop D on the (−) subunit. In α7-AChBP, lobeline pinches onto W53 (loop D), whereas in Aplysia AChBP lobeline adopts an extended binding mode. The dashed line indicates a hydrogen bond.In this study, we took advantage of a recently established α7-nicotinic receptor chimera constructed from the human α7 ligand-binding domain and AChBP (20, 23). This receptor, termed α7-AChBP, shares 71% sequence similarity with the native α7 receptor, including surface-exposed pockets and loops. This is much improved compared with the similarity of 33% between the widely used Aplysia AChBP and the human sequence. Our results show that small amino acid differences result in a different ligand-binding orientation for lobeline, a result critical when performing structure-based drug design. To bias our search toward allosteric binders we used a surface plasmon resonance (SPR)-biosensor-based fragment screening approach in which the orthosteric binding site of α7-AChBP was blocked. This was achieved using two different strategies that were applied in parallel during the screen. The first strategy was to block the orthosteric binding site by preincubation with a high-affinity ligand. The second strategy was to use competition in solution where each fragment was mixed with an orthosteric ligand of lower affinity. This allowed detection of fragments not competing for binding to the orthosteric site and were therefore potential allosteric ligands. Multiple allosteric binders were identified and X-ray crystal structures of α7-AChBP in complex with these fragments are reported herein. These structures reveal three novel allosteric binding sites in the α7 extracellular domain. In combination with electrophysiological recordings on the human α7 nAChR we demonstrate that fragment binding at these sites modulates receptor function. Altogether, the results reveal a molecular blueprint of novel binding sites in the α7 nAChR. This work paves the way for future development of novel positive allosteric modulators of the α7 nAChR, which is intensively studied as a potential target to improve cognitive impairment in certain disease states, including Alzheimer’s disease (1, 24).     

Ventricular volume and neurocognitive outcome after endoscopic third ventriculostomy: is shunting a better option? A review

Background

Shunts are generally associated with a smaller post-treatment ventricular size in comparison to endoscopic third ventriculostomy (ETV).

Methods

To determine whether such a difference in ventricular size has neurocognitive implications, we reviewed the current literature pertaining to the (1) neurocognitive sequelae of hydrocephalus, (2) neurocognitive outcome after ETV, (3) extent of reversal of neurocognitive changes associated with hydrocephalus after shunting, and (4) data on correlation between post-treatment ventricular volume and neurocognitive outcome after ETV.

Results

Collectively, the results of the available studies should call into question the correlation between the residual postoperative ventricular volume and neurocognitive outcome.

Conclusion

The available literature is so far in support of ETV as a valid and effective treatment modality in hydrocephalic patients. No sufficient evidence is available to justify resorting to shunting on the premise that it is associated with a better neurocognitive outcome.

     

The effect of the second coordination sphere on the magnetism of [Ln(NO3)3(H2O)3]·(18-crown-6) (Ln = Dy and Er)

The objective of this work was the exploration of the effect of the second coordination sphere on the magnetic properties of [Ln(NO₃)₃(H₂O)₃]·(18C6) (Ln = Dy (1) and Er (2)) compounds comprising co-crystallized 18-crown-6 ethers. Both compounds were identified as field-induced single molecule magnets (SMMs) with estimated magnetization reversal barriers U_eff = 66–71 K for 1 and U_eff = 21–24 K for 2. Theoretical calculations with the B3LYP functional revealed substantial change and redistribution of the electrostatic potential upon accounting for the second coordination sphere represented by two 18C6 molecules, which resulted in the change of the crystal-field around metal atoms. As a result, the multireference CASSCF calculations exposed significant impact of the second coordination sphere on the energy splitting of the respective ⁶H_15/2 (Dy^III) and ⁴I_15/2 (Er^III) ground states, the magnetization reversal barrier and the magnetic anisotropy parameters. Moreover, the calculated magnetization reversal barriers, U_calc. = 57 K for 1 and U_calc. = 16 K for 2, are in good agreement with the experimental values accentuating the importance of the second coordination sphere on the magnetic properties of SMMs.

The impact of the second coordination sphere on the magnetic properties of [Ln(NO₃)₃(H₂O)₃]·(18C6) compounds comprising co-crystallized 18-crown-6 ethers was investigated.     

Levels of dipeptidyl peptidase IV/CD26 substrates neuropeptide Y and vasoactive intestinal peptide in rheumatoid arthritis patients

Neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) have their biological half-lives controlled by dipeptidyl peptidase IV (DPP IV/CD26). Several lines of evidence suggest the involvement of NPY in the regulation of rheumatoid arthritis (RA), and VIP has already been identified as a potent anti-inflammatory factor that reduces joint inflammation. The role of DPP IV/CD26 in the pathogenesis of RA has been indicated, but its mediator actions involving NPY and VIP have not been well investigated, so the aim of this study was to find an association between NPY, VIP, and DPP IV/CD26 in RA patients. Assessment of NPY, VIP, DPP IV/CD26 as well as some other inflammatory markers was carried out in 20 RA patients being treated with different types of drugs. Control group consisted of 18 osteoarthritis patients. Synovial fluid and serum content of investigated molecules was determined by ELISA and DPP IV/CD26 activity was measured spectrophotometrically. Immunodetection showed elevated levels of NPY and VIP in RA patients, with a significant increase in synovial fluid, while concentration and activity of DPP IV/CD26 were significantly decreased in both synovial fluid and serum. Positive correlations between serum DPP IV/CD26 concentration and activity (R = 0.6961), as well as between serum and synovial fluid concentration of VIP (R = 0.7029) were found. In RA group, NPY, VIP, and DPP IV/CD26 concentrations were not affected by the administration of drugs. The results of this study indicate a connection between elevated concentration of NPY and VIP and decreased DPP IV/CD26 activity and concentration, suggesting a potential role of these molecules in the immunomodulation of RA.     

Asymptomatic atherosclerosis and hypertension in nondiabetic patients with chronic kidney disease

Abstract:  Atherosclerosis is accelerated in dialysis patients, but less is known about asymptomatic atherosclerosis and major risk factors in patients with different stages of chronic kidney disease (CKD). We compared intima media thickness (IMT) and plaque occurrence in the carotid arteries in 104 nondiabetic patients (stages 1–5 of CKD; mean age: 51.6 years) with those in 40 healthy control subjects. The IMT values (0.69 vs. 0.59 mm; P  < 0.002) were higher in patients. More patients had plaques (46.2 vs. 17.5%; P  < 0.002), and number of plaques was higher ( P  < 0.003). Negative correlation between IMT ( P  < 0.0001), presence of plaques ( P  < 0.0001), their number ( P  < 0.040), and chromium 51-labeled ethylenediaminetetraacetate (⁵¹Cr-EDTA) clearance were found in patients. With multiple regression analysis, relationship between IMT and ⁵¹Cr-EDTA clearance ( P  < 0.001) and presence of hypertension ( P  < 0.001) was found. Nondiabetic patients with CKD showed advanced atherosclerosis and IMT, plaque occurrence, and number increased directly with the level of renal dysfunction. Another important risk factor was hypertension.       

Non-insertion-related complications of central venous catheterization--temporary vascular access for hemodialysis

The authors analyzed 309 central venous catheters (CVC) inserted in 147 hemodialysis patients before the maturation of the first or new arteriovenous fistula. One clinical manifestations of sepsis after CVC insertion was found. In all, 33.7% of the catheters were removed because of early minor complications: CVC occlusion, inadequate blood flow in CVC, shattered suture and malposition of CVC, fever, signs of infection at the site of CVC insertion, and bleeding at the site of CVC insertion. The most frequently isolated pathogenic bacteria at the tips of the catheters were coagulase-negative staphylococci highly sensitive to vancomycin and gentamicin.     

Lycopene isomerisation and storage in an in vitro model of murine hepatic stellate cells

Background  Lycopene is a carotenoid whose biological activities and protective effect on prostate and breast cancer have been described, but little is known on its extra-intestinal metabolism and storage. While most alimentary lycopene is in all-trans configuration, in animal and human tissues approximately half of the lycopene is in cis isoforms. Aim of study  Our object was to monitor the capacity of storage, isomerisation, and intracellular localization of all-trans and cis lycopene in hepatic stellate cells, which are the major sites of metabolism and storage of retinoids and carotenoids in the body. Methods  We used the GRX cell line representative of murine hepatic stellate cells, incubated with 1–30 μM lycopene in culture medium. Analysis was done by high-performance liquid chromatography. Results  Lycopene was able to induce expression of the lipocyte phenotype and it was internalized into GRX cells. Its cellular release only occurred in presence of albumin with a rapid initial decrease of intracellular lycopene. A corresponding increase in the culture medium was observed at 24 h. All-trans, 13-cis and 9-cis lycopene isoforms were identified in all the cell compartments. The membrane fraction contained the major part of lycopene, followed by the cytoplasmic fraction, lipid droplets and nuclei. The ratio between all-trans and cis isomers was approximately 2/1 in the majority parts of cell compartments. Conclusions  This study identified a novel hepatic cell type able to store and isomerise lycopene. Liver can contribute to the serum and tissue equilibrium of cis/trans isomers of lycopene, and to participate in storage of lycopene under high extracellular concentration such as observed after the alimentary input.     

Histamine uptake by human endometrial cells expressing the organic cation transporter EMT and the vesicular monoamine transporter-2

Cellular reuptake of monoamines, which is mediated by cell membrane transporters, is followed by accumulation in vesicles by vesicular monoamine transporters (VMAT). The aim of this study was to demonstrate the presence of functional monoamine transporters with high affinity for histamine in human endometrial tissue, since histamine has been implicated as a paracrine signal during endometrial decidualization and embryo implantation. In situ hybridization with (35)S-labelled cRNA probes was used for detection of the organic cationic transporter-2 (OCT-2), the extraneuronal monoamine transporter (EMT), and VMAT-2 in cryosections of normal human endometrial tissue. To identify functional transporters for histamine in endometrial cells, we incubated primary cultures of stromal cells and cultures of attached glands with (3)H-labelled histamine. Cultures were pretreated with either corticosterone, a specific inhibitor of EMT, or reserpine, a specific inhibitor of VMAT-2. EMT mRNA was localized in the stroma with peak expression in the secretory phase, whereas OCT-2 mRNA was expressed by few cells in the stroma throughout the cycle. VMAT-2 mRNA was localized in the stroma during the proliferative phase and in the epithelium during the secretory phase. Thus, EMT and VMAT-2, which both have high affinity for histamine, are strongly expressed in endometrial cells. Both corticosterone and reserpine significantly reduced the uptake of (3)H-histamine in stromal cells during the proliferative as well as the secretory phase. This indicates the presence of functional EMT and VMAT-2 transporter proteins throughout the cycle, even though their periods of maximal mRNA expression were limited. The results of uptake experiments with glandular epithelial cells confirmed not only the presence of functional VMAT-2 transporter protein in the secretory phase but also the absence of a histamine-specific plasma membrane transporter throughout the cycle. Thus, endometrial tissue contains both plasma membrane and vesicular membrane monoamine transporters with high affinity for histamine. They can potentially influence the reproductive process by the uptake of extracellular histamine and subsequent release on demand.