Structure of Native Lens Connexin 46/50 Intercellular Channels by Cryo-EM

Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which—together with computational studies—elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. ‘Hot spots’ of genetic mutations linked to hereditary cataract formation map to the core structural–functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects

Diclofenac and other non-steroidal anti-inflammatory drugs (NSAIDs) are competitive antagonists of the human P2X3 receptor

Introduction: The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is expressed in sensory neurons and involved in nociception. P2X3R inhibition was shown to reduce chronic and neuropathic pain. In a previous screening of 2000 approved drugs, natural products, and bioactive substances, various non-steroidal anti-inflammatory drugs (NSAIDs) were found to inhibit P2X3R-mediated currents.Methods: To investigate whether the inhibition of P2X receptors contributes to the analgesic effect of NSAIDs, we characterized the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp electrophysiology.Results: We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency (with IC50 values of 138.2 and 76.7 µM, respectively). A weaker inhibition of hP2X1R, hP2X4R, and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) inhibited hP2X3R, rP2X3R, and hP2X7R (IC50 values of 221 µM, 264.1 µM, and ∼900 µM, respectively), calling into question its use as a non-selective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of hP2X3R or hP2X2/3R by diclofenac could be overcome by prolonged ATP application or increasing concentrations of the agonist α,β-meATP, respectively, indicating competition of diclofenac and the agonists. Molecular dynamics simulation showed that diclofenac largely overlaps with ATP bound to the open state of the hP2X3R. Our results suggest a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains, inhibits the gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In summary, we demonstrate the inhibition of the human P2X3 receptor by various NSAIDs. Diclofenac proved to be the most effective antagonist with a strong inhibition of hP2X3R and hP2X2/3R and a weaker inhibition of hP2X1R, hP2X4R, and hP2X7R.Discussion: Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac, which are rarely reached in the therapeutic range, may play a minor role in analgesia compared to the high-potency cyclooxygenase inhibition but may explain the known side effect of taste disturbances caused by diclofenac

The Potential of Regional Integration Agreements (RIAs) in Enhancing the Credibility of Reform: The Case of the Syrian-European Association Agreement

The effect of 'locking-in' economic reform and enhancing its credibility is generally regarded as one of the most important potential effects of regional integration. Based on a detailed review of the theoretical debate, this paper develops a general framework for assessment to evaluate the ability of RIAs to serve as effective mechanisms for 'commitment' and 'signalling'. In the second part, this assessment framework is applied to the case of the Syrian-European Association Agreement (AA). Syria initialled an AA with the European Union in October 2004, but two and a half years later, this agreement is still pending formal signature. The empirical findings of this study show that despite several shortcomings, the Syrian-European AA, if it were to come into force, should be able to deliver an appropriate mechanism for signalling and commitment and thus to improve the credibility of the Syrian process of reform at home and abroad. A major loophole of the agreement, however, is represented by its lack of incentives to increase the 'rewards for good policy'.Der Effekt der Verankerung von Reformen und die Erhöhung ihrer Glaubwürdigkeit (Lock-in-Effekt) gilt allgemein als einer der wichtigsten Effekte der regionalen Integration. Aufbauend auf einer detaillierten Betrachtung der theoretischen Debatte zum 'Lock-in-Effekt' wird in diesem Papier ein allgemeiner Analyserahmen entworfen, der dazu dient, die Fähigkeit von RIA als Mechanismen der Signalisierung und Selbstbindung zu bewerten. Im anschließenden zweiten Teil wird dieser Analyserahmen auf das Beispiel des Syrisch-Europäischen Assoziierungsabkommens angewendet. Syrien initialisierte im Oktober 2004 ein Assoziierungsabkommen mit der Europäischen Union. Zweieinhalb Jahre später ist dieses Abkommen noch immer nicht formell unterzeichnet. Die Ergebnisse dieser Studie zeigen, dass das Syrisch-Europäische Assoziierungsabkommen - sollte es in Kraft treten - trotz einiger Mängel einen adäquaten Mechanismus der Signalisierung und Selbstbindung liefern und dazu beitragen kann, die Glaubwürdigkeit des syrischen Reformprozesses in - und außerhalb des Landes zu verbessern. Eine wesentliche Schwäche des Abkommensentwurfs liegt allerdings in mangelnden Anreizen für eine 'Belohnung der guten Politik'

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

Functional Characterization of V-J-gating and Single Channel Conductance of Sheep Cx46 and Cx50 Gap Junctions

Connexins form intercellular communication channels, known as gap junctions (GJs), found throughout vertebrate species (21 isoforms in human). GJs formed by different connexins harbor unique channel properties that have not been fully defined. High-resolution structures of native Cx46 and Cx50 GJs from sheep (sCx46 and sCx50) were recently resolved by single particle CryoEM. The CryoEM based structures of sCx46 and sCx50 were proposed to adopt a more stable open-state conformation, as compared to hCx26, based on structural differences and results of comparative molecular dynamics (MD) studies. These MD-studies identified the NT domains, especially the 9 th position, as key determinants in the differences of energetic barrier to K + permeation in sCx46 and sCx50 GJs. To better align functional studies with the CryoEM structure models, we studied functional properties of GJs formed by sCx46, sCx50, NT domain swapped chimeras (sCx46-50NT and sCx50-46NT), and point variants at the 9 th residue (sCx46-R9N and sCx50-N9R) in GJ-deficient N2A cells. All of them formed functional GJs except sCx46-50NT. Similar Vj-gating properties were observed for sCx46, sCx50, and sCx46-R9N. However, significantly different single channel conductances were observed for these connexin variants. MD-simulations performed on sCx46-R9N and sCx50-N9R show altered free-energy barriers to ion-permeation that are consistent with experimentally obtained single channel conductance. Together, these studies show the NT-domain encodes key functional differences in the rate of ion permeation and V j-gating properties of sCx46 and sCx50 GJs. Supported by NSERC and NIH

A Novel Homozygous KLHL3 Mutation as a Cause of Autosomal Recessive Pseudohypoaldosteronism Type II Diagnosed Late in Life

Introduction: Pseudohypoaldosteronism type II (PHA II) is a Mendelian disorder, featuring hyperkalemic acidosis and low plasma renin levels, typically associated with hypertension. Mutations in WNK1, WNK4, CUL3, and KLHL3 cause PHA II, with dominant mutations in WNK1, WNK4, and CUL3 and either dominant or recessive mutations in KLHL3. Fourteen families with recessive KLHL3 mutations have been reported, with diagnosis at the age of 3 months to 56 years, typically in individuals with normal kidney function. Methods: We performed clinical and genetic investigations in a patient with hyperkalemic hypertension and used molecular dynamics simulations, heterologous expression in COS7 cells, and Western blotting to investigate the effect of a KLHL3 candidate disease mutation on WNK4 protein expression. Results: The patient, a 58-year-old woman from a consanguineous family, showed hypertension, persistent hyperkalemic acidosis associated with severe muscle pain, nephrolithiasis, chronic kidney disease (CKD), and coronary heart disease. Therapy with hydrochlorothiazide corrected hyperkalemia, hypertension, and muscle pain. Genetic analysis revealed a homozygous p.Arg431Trp mutation at a highly conserved KLHL3 position. Simulations suggested reduced stability of the mutant protein, which was confirmed by Western blot. Compared with wild-type KLHL3, cotransfection of p.Arg431Trp KLHL3 led to increased WNK4 protein levels, inferred to cause increased NaCl reabsorption via the thiazide-sensitive carrier and PHA II. Conclusions: Even in patients presenting late in life and in the presence of CKD, PHA II should be suspected if renin levels are low and hyperkalemic acidosis and hypertension are inadequate for CKD stage, particularly in the presence of a suspicious family histor

Connexin 46 and connexin 50 gap junction channel properties are shaped by structural and dynamic features of their N‐terminal domains

Gap junctions formed by different connexins are expressed throughout the body and harbor unique channel properties that have not been fully defined mechanistically. Recent structural studies by Cryo-EM have produced high-resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure-function comparison. Here, we conducted comparative MD simulation and electrophysiology studies to dissect the isoform-specific differences in Cx46 and Cx50 intercellular channel function. We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N-terminal domains, in particular the residue at the 9 position and differences in hydrophobic anchoring sites. The results of this study establish the open state Cx46/50 structural models as archetypes for structure-function studies targeted at elucidating gap junction channel mechanism and the molecular basis of disease-causing variants

Connexin 46 and Connexin 50 Gap Junction Channel Properties Are Shaped by Structural and Dynamic Features of Their N-Terminal Domains.

Gap junctions formed by different connexins are expressed throughout the body and harbor unique channel properties that have not been fully defined mechanistically. Recent structural studies by Cryo-EM have produced high-resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure-function comparison. Here, we conducted comparative MD simulation and electrophysiology studies to dissect the isoform-specific differences in Cx46 and Cx50 intercellular channel function. We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N-terminal domains, in particular the residue at the 9 position and differences in hydrophobic anchoring sites. The results of this study establish the open state Cx46/50 structural models as archetypes for structure-function studies targeted at elucidating gap junction channel mechanism and the molecular basis of disease-causing variants

Visualization of Protein-Lipid Interactions in Connexin-46/50 Intercellular Channels by Cryo-EM and MD-Simulation

Cell-to-cell communication by gap junctions is facilitated by a unique macromolecular architecture, where intercellular channels directly couple the plasma membranes of two neighboring cells. In each membrane, a “hemi-channel” is formed by the oligomerization of six individual subunits (called connexins). To gain insight toward how gap junctions interact with their local membrane environment, we used lipid nanodisc technology to incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid membrane system – closely mimicking a native cell-to-cell junction. Structural characterization of Cx46/50 lipid-embedded channels by single particle CryoEM revealed a drastic lipid-induced stabilization to the channel architecture, resulting in a 3D reconstruction at 2.1 Å resolution – providing an unprecedented level of detail for the class of protein. The subunit packing within each hemi-channel is buttressed by a bouquet of highly-stabilized lipid acyl-chains. Remarkably, the stabilizing effects to the lipid-bilayer extends well beyond the annular layer of lipids and effectively immobilizes the extra-cellular leaflets of the two connected membranes. In addition, over 150 water molecules are resolved within the channel architecture. Time-averaged densities of the lipids and water obtained by MD simulation display the same pattern of stabilization seen in the CryoEM reconstruction. MD analysis suggest lipid headgroups remain highly dynamic relative to the lipid acyl-chains, rationalizing the absence of headgroup densities in the 2.1 Å CryoEM density map. 3D heterogeneity analysis of the CryoEM data identified 4 distinct classes of lipid headgroup configurations, which were also detected by network analysis of extracellular lipids in MD-simulations. Lipid and water binding sites identified in Cx46/50 are remarkably conserved across the connexin family, suggesting these stabilizing features are key contributors to the structure and function of gap junction intercellular communication