Hydrophobic residues at position 10 of α-conotoxin PnIA influence subtype selectivity between α7 and α3β2 neuronal nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer's disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3β2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3β2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki = 44 nM) though selectivity reverted to α3β2 (IC50 = 0.7 nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3β2 nAChRs via different molecular determinants

Single amino acid substitutions in alpha-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor

The alpha-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication, In this study, the native alpha-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the ACh-induced current in all rat parasympathetic neurons examined, with IC50 values of 14 and 33 nM, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified alpha-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC50 value of 375 nM and a maximally effective concentration caused 91% block, [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in similar to 80% of neurons, with an IC50 value of 1.4 nM and 46% maximal block of the total current, The residual current was not inhibited further by alpha-bungarotoxin, but was further reduced by the cu-conotoxins PnIA or PnIB, and by mecamylamine. H-1 NMR studies indicate that PnIA, PnIB, and the analogues, [A10L]PnIA and [N11S]PnIA, have identical backbone structures. We propose that positions 10 and II of PnIA and PnIB influence potency and determine selectivity among alpha 7 and other nAChR subtypes, including alpha 3 beta 2 and alpha 3 beta 4, Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conopeptides

The Milky Way Tomography with SDSS: III. Stellar Kinematics

We study Milky Way kinematics using a sample of 18.8 million main-sequence stars with r<20 and proper-motion measurements derived from SDSS and POSS astrometry, including ~170,000 stars with radial-velocity measurements from the SDSS spectroscopic survey. Distances to stars are determined using a photometric parallax relation, covering a distance range from ~100 pc to 10 kpc over a quarter of the sky at high Galactic latitudes (|b|>20 degrees). We find that in the region defined by 1 kpc <Z< 5 kpc and 3 kpc <R< 13 kpc, the rotational velocity for disk stars smoothly decreases, and all three components of the velocity dispersion increase, with distance from the Galactic plane. In contrast, the velocity ellipsoid for halo stars is aligned with a spherical coordinate system and appears to be spatially invariant within the probed volume. The velocity distribution of nearby ($Z<1$ kpc) K/M stars is complex, and cannot be described by a standard Schwarzschild ellipsoid. For stars in a distance-limited subsample of stars (<100 pc), we detect a multimodal velocity distribution consistent with that seen by HIPPARCOS. This strong non-Gaussianity significantly affects the measurements of the velocity ellipsoid tilt and vertex deviation when using the Schwarzschild approximation. We develop and test a simple descriptive model for the overall kinematic behavior that captures these features over most of the probed volume, and can be used to search for substructure in kinematic and metallicity space. We use this model to predict further improvements in kinematic mapping of the Galaxy expected from Gaia and LSST.Comment: 90 pages, 26 figures, submitted to Ap

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.This study, including the enrollment of cases, sequencing, and analysis received support from the National Institute for Health Research (NIHR) BioResource–Rare Diseases. The NIHR BioResource is funded by the NIHR (http://www.nihr.ac.uk). Research in the Ouwehand Laboratory is also supported by grants from Bristol-Myers Squibb, the British Heart Foundation, the British Society of Haematology, the European Commission, the MRC, the NIHR, and the Wellcome Trust; the laboratory also receives funding from National Health Service Blood and Transplant (NHSBT). The clinical fellows received funding from the MRC (C.L. and S.K.W.); the NIHR–Rare Diseases Translational Research Collaboration (S. Sivapalaratnam); and the British Society for Haematology and National Health Service Blood and Transplant (T.K.B.).This is the author accepted manuscript. The final version is available from American Society of Hematology via http://dx.doi.org/10.1182/blood-2015-12-688267

An ATP-sensitive K+ conductance in dissociated neurones from adult rat intracardiac ganglia

An ATP-sensitive K+ (KATP) conductance has been identified using the perforated patch recording configuration in a population (52%) of dissociated neurones from adult rat intracardiac ganglia. The presence of the sulphonylurea receptor in approximately half of the intracardiac neurones was confirmed by labelling with fluorescent glibenclamide-BODIPY.Under current clamp conditions in physiological solutions, levcromakalim (10 μm) evoked a hyperpolarization, which was inhibited by the sulphonylurea drugs glibenclamide and tolbutamide.Under voltage clamp conditions in symmetrical (140 mm) K+ solutions, bath application of levcromakalim evoked an inward current with a density of ∼8 pA pF−1 at –50 mV and a slope conductance of ∼9 nS, which reversed close to the potassium equilibrium potential (EK). Cell dialysis with an ATP-free intracellular solution also evoked an inward current, which was inhibited by tolbutamide.Bath application of either glibenclamide (10 μm) or tolbutamide (100 μm) depolarized adult intracardiac neurones by 3–5 mV, suggesting that a KATP conductance is activated under resting conditions and contributes to the resting membrane potential.Activation of a membrane current by levcromakalim was concentration dependent with an EC50 of 1.6 μm. Inhibition of the levcromakalim-activated current by glibenclamide was also concentration dependent with an IC50 of 55 nm.Metabolic inhibition with 2,4-dinitrophenol and iodoacetic acid or superfusion with hypoxic solution (PO2∼16 mmHg) also activated a membrane current. These currents exhibited similar I–V characteristics to the levcromakalim-induced current and were inhibited by glibenclamide.Activation of KATP channels in mammalian intracardiac neurones may contribute to changes in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion

Ciguatoxin (CTX-1) modulates single tetrodotoxin-sensitive sodium channels in rat parasympathetic neurones

The actions of the marine neurotoxin, ciguatoxin-1 (CTX-1), were investigated in isolated parasympathetic neurones from neonatal rat intracardiac ganglia using patch-clamp recording techniques. Under current clamp conditions, bath application of 1-10 nM CTX-1 caused gradual membrane depolarization and tonic action potential firing. Action potential firing ceased with depolarization beyond approximately -35 mV and application of 300 nM tedrodotoxin (TTX) repolarized the cell to its control resting potential. In cell-attached membrane patches, 1-10 nM CTX-1 in the patch pipette markedly increased the open probability of single TTX-sensitive Na+ channels in response to depolarizing voltage steps but did not alter the unitary conductance (10 pS) or reversal potential. Under steady-state conditions, CTX-1 caused spontaneous opening of single Na+ channels which did not inactivate at hyperpolarized membrane potentials. CTX-1 increases neuronal excitability by shifting the voltage of activation of TTX-sensitive Na+ channels to more negative potentials

An automated system for intracellular and intranuclear injection

The Xenopus oocyte expression system has played an important role in the study of cellular proteins, particularly in the field of membrane physiology; expression of transporters and ion channels has significantly advanced our knowledge of these membrane proteins and the rapid and easy expression of mutants has been crucial in many structure-function studies. Xenopus oocytes are an expression system in many ligand-binding assays and in functional screening for ion channel modulators. Several commercially available automated technologies use this system, generating a demand for large numbers of oocytes injected with ion channel genes. Injection of oocytes with genetic material is generally carried out manually. Here we describe an automated system capable of injecting up to 600 oocytes per hour. Oocytes are contained in microplates with conical wells, a simple calibration procedure by the operator is required and pipette filling and oocyte injection are carried out automatically. Following intracellular injection of mRNA coding for ligand-gated ion channels close to 100% of oocytes tested positive for expression, and intranuclear injection of cDNA gave a rate of expression >50%. Moreover, we demonstrate that this method can also be successfully applied to inject zebrafish embryos and could be extended to other cell types

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+. the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207±19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 μM and slope of ≅3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 μM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i \u3c100 nM and the increased channel activity evoked by ionomycin was consistent with a rise in [Ca2+]i to ≥ 0.3 μM. TEA (0.2-1 mM) increased the action potential duration 1.5-fold and reduced the amplitude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP amplitude but reduced the AHP duration by 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons