Mechanism of Ivermectin Facilitation of Human P2X4 Receptor Channels

Ivermectin (IVM), a widely used antiparasitic agent in human and veterinary medicine, was recently shown to augment macroscopic currents through rat P2X4 receptor channels (Khakh, B.S., W.R. Proctor, T.V. Dunwiddie, C. Labarca, and H.A. Lester. 1999. J. Neurosci. 19:7289–7299.). In the present study, the effects of IVM on the human P2X4 (hP2X4) receptor channel stably transfected in HEK293 cells were investigated by recording membrane currents using the patch clamp technique. In whole-cell recordings, IVM (≤10 μM) applied from outside the cell (but not from inside) increased the maximum current activated by ATP, and slowed the rate of current deactivation. These two phenomena likely result from the binding of IVM to separate sites. A higher affinity site (EC50 0.25 μM) increased the maximal current activated by saturating concentrations of ATP without significantly changing the rate of current deactivation or the EC50 and Hill slope of the ATP concentration-response relationship. A lower affinity site (EC50 2 μM) slowed the rate of current deactivation, and increased the apparent affinity for ATP. In cell-attached patch recordings, P2X4 receptor channels exhibited complex kinetics, with multiple components in both the open and shut distributions. IVM (0.3 μM) increased the number of openings per burst, without significantly changing the mean open or mean shut time within a burst. At higher concentrations (1.5 μM) of IVM, two additional open time components of long duration were observed that gave rise to long-lasting bursts of channel activity. Together, the results suggest that the binding of IVM to the higher affinity site increases current amplitude by reducing channel desensitization, whereas the binding of IVM to the lower affinity site slows the deactivation of the current predominantly by stabilizing the open conformation of the channel

Distinct Axonemal Processes Underlie Spontaneous and Stimulated Airway Ciliary Activity

Cilia are small organelles protruding from the cell surface that beat synchronously, producing biological transport. Despite intense research for over a century, the mechanisms underlying ciliary beating are still not well understood. Even the nature of the cytosolic molecules required for spontaneous and stimulated beating is debatable. In an effort to resolve fundamental questions related to cilia beating, we developed a method that integrates the whole-cell mode of the patch-clamp technique with ciliary beat frequency measurements on a single cell. This method enables to control the composition of the intracellular solution while the cilia remain intact, thus providing a unique tool to simultaneously investigate the biochemical and physiological mechanism of ciliary beating. Thus far, we investigated whether the spontaneous and stimulated states of cilia beating are controlled by the same intracellular molecular mechanisms. It was found that: (a) MgATP was sufficient to support spontaneous beating. (b) Ca2+ alone or Ca2+-calmodulin at concentrations as high as 1 μM could not alter ciliary beating. (c) In the absence of Ca2+, cyclic nucleotides produced a moderate rise in ciliary beating while in the presence of Ca2+ robust enhancement was observed. These results suggest that the axonemal machinery can function in at least two different modes

Secondary Structure and Gating Rearrangements of Transmembrane Segments in Rat P2X4 Receptor Channels

P2X receptors are cation selective channels that are activated by extracellular nucleotides. These channels are likely formed by three identical or related subunits, each having two transmembrane segments (TM1 and TM2). To identify regions that undergo rearrangement during gating and to probe their secondary structure, we performed tryptophan scanning mutagenesis on the two putative TMs of the rat P2X4 receptor channel. Mutant channels were expressed in Xenopus oocytes, concentration–response relationships constructed for ATP, and the EC50 estimated by fitting the Hill equation to the data. Of the 22 mutations in TM1 and 24 in TM2, all but one in TM1 and seven in TM2 result in functional channels. Interestingly, the majority of the functional mutants display an increased sensitivity to ATP, and in general these perturbations are more pronounced for TM2 when compared with TM1. For TM1 and for the outer half of TM2, the perturbations are consistent with these regions adopting α-helical secondary structures. In addition, the greatest perturbations in the gating equilibrium occur for mutations near the outer ends of both TM1 and TM2. Surface biotinylation experiments reveal that all the nonfunctional mutants traffic to the surface membrane at levels comparable to the WT channel, suggesting that these mutations likely disrupt ion conduction or gating. Taken together, these results suggest that the outer parts of TM1 and TM2 are helical and that they move during activation. The observation that the majority of nonconducting mutations are clustered toward the inner end of TM2 suggests a critical functional role for this region

Intracellular Ca2+ Regulates the Phosphorylation and the Dephosphorylation of Ciliary Proteins Via the NO Pathway

The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO syntase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating

Ion access pathway to the transmembrane pore in P2X receptor channels

P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function

Reporting animal research:Explanation and elaboration for the ARRIVE guidelines 2.0

Improving the reproducibility of biomedical research is a major challenge. Transparent and accurate reporting is vital to this process; it allows readers to assess the reliability of the findings and repeat or build upon the work of other researchers. The ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) were developed in 2010 to help authors and journals identify the minimum information necessary to report in publications describing in vivo experiments. Despite widespread endorsement by the scientific community, the impact of ARRIVE on the transparency of reporting in animal research publications has been limited. We have revised the ARRIVE guidelines to update them and facilitate their use in practice. The revised guidelines are published alongside this paper. This explanation and elaboration document was developed as part of the revision. It provides further information about each of the 21 items in ARRIVE 2.0, including the rationale and supporting evidence for their inclusion in the guidelines, elaboration of details to report, and examples of good reporting from the published literature. This document also covers advice and best practice in the design and conduct of animal studies to support researchers in improving standards from the start of the experimental design process through to publication

A call for transparent reporting to optimize the predictive value of preclinical research

The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress

Global, regional, and national disability-adjusted life-years (DALYs) for 333 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016

BACKGROUND: Measurement of changes in health across locations is useful to compare and contrast changing epidemiological patterns against health system performance and identify specific needs for resource allocation in research, policy development, and programme decision making. Using the Global Burden of Diseases, Injuries, and Risk Factors Study 2016, we drew from two widely used summary measures to monitor such changes in population health: disability-adjusted life-years (DALYs) and healthy life expectancy (HALE). We used these measures to track trends and benchmark progress compared with expected trends on the basis of the Socio-demographic Index (SDI). METHODS: We used results from the Global Burden of Diseases, Injuries, and Risk Factors Study 2016 for all-cause mortality, cause-specific mortality, and non-fatal disease burden to derive HALE and DALYs by sex for 195 countries and territories from 1990 to 2016. We calculated DALYs by summing years of life lost and years of life lived with disability for each location, age group, sex, and year. We estimated HALE using age-specific death rates and years of life lived with disability per capita. We explored how DALYs and HALE differed from expected trends when compared with the SDI: the geometric mean of income per person, educational attainment in the population older than age 15 years, and total fertility rate. FINDINGS: The highest globally observed HALE at birth for both women and men was in Singapore, at 75·2 years (95% uncertainty interval 71·9-78·6) for females and 72·0 years (68·8-75·1) for males. The lowest for females was in the Central African Republic (45·6 years [42·0-49·5]) and for males was in Lesotho (41·5 years [39·0-44·0]). From 1990 to 2016, global HALE increased by an average of 6·24 years (5·97-6·48) for both sexes combined. Global HALE increased by 6·04 years (5·74-6·27) for males and 6·49 years (6·08-6·77) for females, whereas HALE at age 65 years increased by 1·78 years (1·61-1·93) for males and 1·96 years (1·69-2·13) for females. Total global DALYs remained largely unchanged from 1990 to 2016 (-2·3% [-5·9 to 0·9]), with decreases in communicable, maternal, neonatal, and nutritional (CMNN) disease DALYs offset by increased DALYs due to non-communicable diseases (NCDs). The exemplars, calculated as the five lowest ratios of observed to expected age-standardised DALY rates in 2016, were Nicaragua, Costa Rica, the Maldives, Peru, and Israel. The leading three causes of DALYs globally were ischaemic heart disease, cerebrovascular disease, and lower respiratory infections, comprising 16·1% of all DALYs. Total DALYs and age-standardised DALY rates due to most CMNN causes decreased from 1990 to 2016. Conversely, the total DALY burden rose for most NCDs; however, age-standardised DALY rates due to NCDs declined globally. INTERPRETATION: At a global level, DALYs and HALE continue to show improvements. At the same time, we observe that many populations are facing growing functional health loss. Rising SDI was associated with increases in cumulative years of life lived with disability and decreases in CMNN DALYs offset by increased NCD DALYs. Relative compression of morbidity highlights the importance of continued health interventions, which has changed in most locations in pace with the gross domestic product per person, education, and family planning. The analysis of DALYs and HALE and their relationship to SDI represents a robust framework with which to benchmark location-specific health performance. Country-specific drivers of disease burden, particularly for causes with higher-than-expected DALYs, should inform health policies, health system improvement initiatives, targeted prevention efforts, and development assistance for health, including financial and research investments for all countries, regardless of their level of sociodemographic development. The presence of countries that substantially outperform others suggests the need for increased scrutiny for proven examples of best practices, which can help to extend gains, whereas the presence of underperforming countries suggests the need for devotion of extra attention to health systems that need more robust support. FUNDING: Bill & Melinda Gates Foundation