Expression, purification, electron microscopy, N-glycosylation mutagenesis and molecular modeling of human P2X4 and Dictyostelium discoideum P2XA

The recent publication of the apo-, closed-state 3D crystal structure of zebrafish (zf) P2X4.1 has not only revolutionized the P2X research field, but also highlighted the need for further crystal structures, of receptors in different activation states, so that we can gain a complete molecular understanding of ion channel function. zfP2X4.1 was selected as a 3D-crystallization candidate because of its ability to form stable trimers in detergent solution, and purified from over-expression in baculovirus-infected Spodoptera frugiperda (Sf9) insect cells. In this work, we have used a similar approach to express both human P2X4 (hP2X4) and Dictyostelium discoideum P2XA (DdP2XA) in Sf9 cells. Although hP2X4 did not form stable trimers in detergent solution, both receptors bound to ATP-coupled resins, indicating that their extracellular domains were folded correctly. DdP2XA formed strong trimers in detergent solution, and we were able to selectively purify trimers using preparative electrophoresis, and build a 21 Å-resolution 3D structure using transmission electron microscopy and single particle analysis. Although the structure of DdP2XA possessed similar dimensions to those of the previously determined low-resolution hP2X4 structure and the zfP2X4.1 crystal structure, N-glycosylation mutagenesis and molecular modeling indicated differences between N-glycan usage and predicted accessibility in models of DdP2XA based on the zfP2X4.1 crystal structure. Our data demonstrate that DdP2XA expressed in insect cells retains ATP-binding capacity after detergent solubilization, is an ideal candidate for structural study, and possesses a significantly different 3D structure to that of both hP2X4 and zfP2X4.1

P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers

The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7

Development and validation of the Japanese version of the Bedtime Procrastination Scale (BPS-J)

Abstract Background The average sleep duration of Japanese people is shorter than that of people from other countries, and bedtime procrastination is suspected to be one of the factors contributing to this issue. This study aimed to develop and validate the Japanese version of the Bedtime Procrastination Scale (BPS-J). Methods The BPS-J was developed through procedures including the translation and back-translation of the scale, cognitive interviews with 100 participants who reported having experiences of being diagnosed with insufficient sleep syndrome (ISS) or receiving treatment for ISS using open-ended online questionnaires, and expert checking. To investigate the scale’s validity and reliability, an online survey was conducted with daytime workers aged 20 − 65 years without a history of sleep disorders other than ISS. Half the participants were retested using the same survey after 14 days. Participants’ responses to the Brief Self-Control Scale (BSCS), General Procrastination Scale (GPS), and Munich ChronoType Questionnaire (MCTQ), and data on sleep-related variables such as sleep duration on workdays and the days per week of fatigue or sleep loss, sex, and age, were collected. Results We analyzed data from 574 participants to assess scale validity. We then analyzed data from 280 participants to determine test–retest reliability. Confirmatory factor analyses revealed that the two-factor model without Item 2 was most suitable for the BPS-J, unlike other language versions. Regardless of the full-item model or the model with Item 2 eliminated, sufficient reliability and significant correlations with the BSCS, GPS, MCTQ, and sleep-related variables such as sleep duration per night on work days, days per week of feeling fatigued, and days per week of sleep loss were observed. Logistic and linear regressions showed that the relationships between the BPS-J, sleep-related variables, and MCTQ were maintained after adjusting for sex and age. Conclusion The BPS-J had sufficient validity and reliability. Further, eliminating Item 2 from the original version of the BPS strengthened the ability to survey Japanese daytime workers

Structure of P2X receptors

P2X receptors are trimeric ion channels that open in response to extracellular ATP. The publication of the first X-ray structure of a closed P2X receptor has confirmed many of the functional experiments carried out since the mid-1990s and provides a point of reference for the molecular dissection of P2X receptor function. These co-ordinates shed light on the ATP binding sites housed in the extracellular domain, the contacts between subunits, the pathways for ion access, and the cation-selective pore that spans the cell membrane. They also provide a template for structure-based design of much needed pharmacological agents to act on a receptor superfamily whose physiological roles extend from synaptic transmission to inflammation and control of programmed cell death. Interpretation of functional data in the context of this closed P2X receptor structure reveals structural rearrangements in the transmembrane pore and extracellular domain. Here the current understanding of the molecular structure of P2X receptors is reviewed. This review will be of relevance to those interested in the mechanisms that underlie the molecular operation of P2X receptors, and other ion channels gated by diffusible ligands (pentameric Cys-loop receptors, tetrameric glutamate receptors), as well as trimeric ion channels that are distantly related to P2X receptors (acid-sensing ion channels and the epithelial sodium channel). © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.