Neuronal junctophilins recruit specific Cav and RyR isoforms to ER-PM junctions and functionally alter Cav2.1 and Cav2.2

Junctions between the endoplasmic reticulum and plasma membrane that are induced by the neuronal junctophilins are of demonstrated importance, but their molecular architecture is still poorly understood and challenging to address in neurons. This is due to the small size of the junctions and the multiple isoforms of candidate junctional proteins in different brain areas. Using colocalization of tagged proteins expressed in tsA201 cells, and electrophysiology, we compared the interactions of JPH3 and JPH4 with different calcium channels. We found that JPH3 and JPH4 caused junctional accumulation of all the tested high-voltage-activated CaV isoforms, but not a low-voltage-activated CaV. Also, JPH3 and JPH4 noticeably modify CaV2.1 and CaV2.2 inactivation rate. RyR3 moderately colocalized at junctions with JPH4, whereas RyR1 and RyR2 did not. By contrast, RyR1 and RyR3 strongly colocalized with JPH3, and RyR2 moderately. Likely contributing to this difference, JPH3 binds to cytoplasmic domain constructs of RyR1 and RyR3, but not of RyR2

Junctophilins 1, 2, and 3 all support voltage-induced Ca2+ release despite considerable divergence

In skeletal muscle, depolarization of the plasma membrane (PM) causes conformational changes of the calcium channel CaV1.1 that then activate RYR1 to release calcium from the SR. Being independent of extracellular calcium entry, this process is termed voltage-induced calcium release. In skeletal muscle, junctophilins (JPHs) 1 and 2 form the SR–PM junctions at which voltageinduced calcium release occurs. Previous work demonstrated that JPH2 is able to recapitulate voltage-induced calcium release when expressed in HEK293 cells together with CaV1.1, β1a, Stac3, and RYR1. However, it is unknown whether JPH1 and the more distantly related neuronal JPH3 and JPH4 might also function in this manner, a question of interest because different JPH isoforms diverge in their interactions with RYR1. Here, we show that, like JPH2, JPH1 and JPH3, coexpressed with CaV1.1, β1a, Stac3, and RYR1 in HEK293 cells, cause colocalization of CaV1.1 and RYR1 at ER–PM junctions. Furthermore, potassium depolarization elicited cytoplasmic calcium transients in cells in which WT CaV1.1 was replaced with the calcium impermeant mutant CaV1.1(N617D), indicating that JPH1, JPH2, and JPH3 can all support voltage-induced calcium release, despite sequence divergence and differences in interaction with RYR1. Conversely, JPH4-induced ER–PM junctions contain CaV1.1 but not RYR1, and cells expressing JPH4 are unable to produce depolarization-induced calcium transients. Thus, JPHs seem to act primarily to form ER–PM junctions and to recruit the necessary signaling proteins to these junctions but appear not to be directly involved in the functional interactions between these proteins

De novo reconstitution reveals the proteins required for skeletal muscle voltage-induced Ca2+ release

Skeletal muscle contraction is triggered by Ca2+ release from the sarcoplasmic reticulum (SR) in response to plasma membrane (PM) excitation. In vertebrates, this depends on activation of the RyR1 Ca2+ pore in the SR, under control of conformational changes of CaV1.1, located ∼12 nm away in the PM. Over the last ∼30 y, gene knockouts have revealed that CaV1.1/RyR1 coupling requires additional proteins, but leave open the possibility that currently untested proteins are also necessary. Here, we demonstrate the reconstitution of conformational coupling in tsA201 cells by expression of CaV1.1, β1a, Stac3, RyR1, and junctophilin2. As in muscle, depolarization evokes Ca2+ transients independent of external Ca2+ entry and having amplitude with a saturating dependence on voltage. Moreover, freeze-fracture electron microscopy indicates that the five identified proteins are sufficient to establish physical links between CaV1.1 and RyR1. Thus, these proteins constitute the key elements essential for excitation-contraction coupling in skeletal muscle

Stac Proteins Suppress Ca2+-Dependent Inactivation of Neuronal L-type Ca2+ Channels

Stac protein (named for its SH3-and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca2+ channels. Overexpression of the three Stac isoforms eliminated Ca2+-dependent inactivation (CDI) ofL-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-L-type current. Using heterologous expression in tsA201 cells (together with β and α2-δ1 auxiliary subunits), we found that CDI for CaV1.2 and CaV1.3 (the predominant, neuronalL-type Ca2+ channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, CaV2.1, was not. For CaV1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel’s C terminus. Within the Stac proteins, a weakly conserved segment containing ~100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI forL-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca2+ entry via neuronal L-type channels. © 2018 the authors

'Here be dragons, here be savages, here be bad plumbing: Australian media representations of sport and terrorism

As 'Propaganda Theorists argue, an examination of key discourses can enhance our understanding of how economic, political and social debate is shaped by mainstream media reporting. In this essay we present content and discourse analysis of Australian media reporting on the nexus of sport and terrorism. Examining newspaper reports over a five-year period, from 1996-2001, which included the 11 September 2001 terrorist tragedy in the United States (9/11), provides useful insights into how public discourse might be influenced with regard to sport and terrorism interrelationships. The results of the media analysis suggest that hegemonic tropes are created around sport and terrorism. The distilled message is one of good and evil, with homilies of sport employed in metaphors for western society and its values. The reactions and responses of sport administrators and athletes to terrorist acts and the threat of terrorism to sport are used to exemplify these ideals, providing newspaper readers a context within which to localize meaning and relevance

Radiographers supporting radiologists in the interpretation of screening mammography: a viable strategy to meet the shortage in the number of radiologists.

BackgroundAn alternative approach to the traditional model of radiologists interpreting screening mammography is necessary due to the shortage of radiologists to interpret screening mammograms in many countries.MethodsWe evaluated the performance of 15 Mexican radiographers, also known as radiologic technologists, in the interpretation of screening mammography after a 6 months training period in a screening setting. Fifteen radiographers received 6 months standardized training with radiologists in the interpretation of screening mammography using the Breast Imaging Reporting and Data System (BI-RADS) system. A challenging test set of 110 cases developed by the Breast Cancer Surveillance Consortium was used to evaluate their performance. We estimated sensitivity, specificity, false positive rates, likelihood ratio of a positive test (LR+) and the area under the subject-specific Receiver Operating Characteristic (ROC) curve (AUC) for diagnostic accuracy. A mathematical model simulating the consequences in costs and performance of two hypothetical scenarios compared to the status quo in which a radiologist reads all screening mammograms was also performed.ResultsRadiographer's sensitivity was comparable to the sensitivity scores achieved by U.S. radiologists who took the test but their false-positive rate was higher. Median sensitivity was 73.3 % (Interquartile range, IQR: 46.7-86.7 %) and the median false positive rate was 49.5 % (IQR: 34.7-57.9 %). The median LR+ was 1.4 (IQR: 1.3-1.7 %) and the median AUC was 0.6 (IQR: 0.6-0.7). A scenario in which a radiographer reads all mammograms first, and a radiologist reads only those that were difficult for the radiographer, was more cost-effective than a scenario in which either the radiographer or radiologist reads all mammograms.ConclusionsGiven the comparable sensitivity achieved by Mexican radiographers and U.S. radiologists on a test set, screening mammography interpretation by radiographers appears to be a possible adjunct to radiologists in countries with shortages of radiologists. Further studies are required to assess the effectiveness of different training programs in order to obtain acceptable screening accuracy, as well as the best approaches for the use of non-physician readers to interpret screening mammography